Sample records for raji cell assay

  1. The Effect of Overexpression of hTERT on Etoposide (VP-16)-Induced Apoptosis in Raji Cells

    Dongmei He; Huan Zhang; Gexiu Liu; Wei Yu


    OBJECTIVE To explore whether overexpression of human telomerase re-verse transcriptase (hTERT) in Raji cells can protect against etoposide (VP-16)-induced apoptosis.METHODS A lipofectin-mediated gene transfection method was used to transfer the hTERT gene into Raji cells. The polymerase chain reaction enzyme-linked immunoassay was employed to determine telomerase activity.The expression levels of hTERT protein were assayed by immunofluorescence using a fluoresce isothiocyanate label. Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was assessed by morphological observation and flow cytometry analysis.RESULTS The results showed that there was a marked increase in both mean fluorescence intensity of hTERT-protein-positive cells and telomerase activity in hTERT- transfected Raji cells (P <0.05), but there was no difference in hTERT protein and telomerase activity levels between Raji cells and vectortransfected Raji cells (P >0.05). There were more viable cells at 48 h and 72 h after treatment of hTERT-transfected Raji cells with 10 μmol/L VP-16 compared to either vector-transfected Raji cells and Raji cells (P <0.05). Apoptosis rates at 72 h after treatment with 10 μmol/L VP-16 were 4.34 ±1.03% in hTERT-transfected Raji cells, 33.21 ± 3.12% in vector-transfected Raji cells,and 31.63 ± 3.06% in Raji ceils. There was a significant difference in the percentage of apoptotic cells between hTERT-transfected Raji cells and either vector-transfected Raji cells or Raji cells (P<0.05).CONCLUSION Overexpression of telomerase by transfection of hTERT gene can protect against etoposide-induced apoptosis in Raji cells.

  2. Effects of Triptolide on Cell Proliferation and CXCR4 Expression in Burkitt's Lymphoma Raji Cells In Vitro

    ZHANG Chun; CUI Guo-hui; LIU Fang; WU Qiu-ling; CHEN Yan


    Objective: To investigate the inhibitory effects of triptolide on cell proliferation and CXCR4 expression in Burkitt's lymphoma cell line Raji cells. Methods: The effects of triptolide on the growth of Raji cells were studied by 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium(MTT) assay. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α (rhSDF-1α) in vitro. Results: Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. Triptolide could downregulate the CXCR4 expression on Raji cells in a dose-dependent manner. Furthermore, chemotaxis assays showed that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent. Conclusion: Triptolide could inhibit the proliferation and migration of Raji cells in vitro. The underlying anti-tumor mechanism of triptolide might be related to the anti-proliferative effect and the blockage of SDF-1/CXCR4 axis.


    CHEN Yan; WU Qing; LI Xin-gang


    Objective: To study the growth-inhibitory effects of curcumin on B-NHL cell line Raji cells in vitro and its molecular mechanisms. Methods: The growth inhibition rates of Raji cells, after being treated with 6.25 μmol/L~50 μmol/L curcumin for 12 h ~ 48 h, were examined by MTT assay. The apoptosis rate was detected by flow cytometry (FCM), the protein expression levels of bcl-2 and p53 in Raji cells were examined by SP immunohistochemistry. The expression of p53 in Raji cell were checked by RT-PCR. Results: After being treated by various concentrations of curcumin, the growth of Raji cells was inhibited significantly. The rates of apoptosis were 11.8% ~79.7% (P<0.01), the down regulation of p53 expression was observed within 24 h after the treatment of curcumin by RT-PCR. The expression of bcl-2 and p53 was decreased, which depended on the action time. Conclusion: Curcumin could significantly inhibit the growth of Raji cells. The induction of apoptosis by down-regulating the expression of bcl-2 and p53 was probably one of its molecular mechanisms.

  4. Apoptosis in Raji cell line induced by influenza A virus

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远


    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  5. Hexa-arginine enhanced uptake and residualization of selective high affinity ligands by Raji lymphoma cells

    Mirick Gary


    Full Text Available Abstract Background A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL (DvLPBaPPP2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. Results An analog of the SHAL (DvLPBaPPP2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. Conclusion The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a

  6. Studies of Liposomal bcl-2 Antisense Oligode-oxynucleofide Induction of Apoptosis in Raji Cells

    DongmeiHe; HuanZhong


    OBJECTIVE To explore the effect of liposomal G3139 and transfected antisense phosphorothioate oligodeoxynucleotides directed against the coding region of the bcl-2 messenger RNA and the translation site on apoptosis in Raji cells.METHODS Cytotoxic effects were measured by use of the MTT method; The expression levels of Bcl-2 protein were assayed by immunofiuorescence using a fluoresce isothiocyanate label. Apoptosis was determined by morphological observation and flow cytometric analysis.RESULTS The 2 antisense oligonucleotides and G3139 can reduce Bcl-2 protein levels and Raji cell viability (IC50=4.54, 4.72 and 4.26 μmol/L, respectively), and induce apoptosis. A scrambled sequence control oligonucleotide and empty liposomes did not alter cell viability, Bcl-2 protein expression or apoptosis rates. There was no difference in reducing Bcl-2 protein levels and apoptosis rates found among the 3 antisense oligonucleotides.CONCLUSION The 2 antisense oligodeoxynucleotides of bcl-2 messenger RNA can effectively induce apoptosis of Raji cells. The 2 antisense sequences and G3139 have a similarity in their antisense effect.

  7. Curcumin, a potent anti-tumor reagent, is a novel histone deacetylase inhibitor regulating B-NHL cell line Raji proliferation

    Hong-li LIU; Yan CHEN; Guo-hui CUI; Jian-feng ZHOU


    Aim: To investigate curcumin (diferuloylmethane) induced apoptosis and its molecular mechanism of action in B-NHL cell line Raji cells. Methods: Raji cells were cultured in RPMI- 1640 medium and treated with curcumin in different concentrations. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay was used to detect growth inhibition and Hoechst 33258 staining was used to detect apoptosis. Immunocytochemistry and Western blot were used to detect the expressions of histone deacetylase 1, 3, and 8 (HDAC 1, HDAC3, and HDAC8) and acetylated histone H4 (Ac-histone H4) protein. Results: Curcumin inhibited the proliferation of B-NHL cell line Raji cells with a 36-h IC50 value of 24.1±2.0 μmol/L.Hoechst 33258 staining showed that curcumin could induce Raji cell apoptosis.The expression levels of HDAC 1, HDAC3, and HDAC8 proteins were downregulated following curcumin treatment in Raji cells, whereas Ac-histone H4 protein expression was upregulated after treatment with curcumin. Conclusion: Curcumin, as a new member of the histone deacetylase inhibitors, can inhibit the expression of class I HDACs (HDAC1, HDAC3, and HDAC8), and can increase the expression of Ac-histone H4 in Raji cells. Curcumin plays an important role in regulating B-NHL cell line Raji cell proliferation and apoptosis.


    HE Dong-mei; ZHANG Huan


    Objective: To investigate whether the Bc1-2 antisense oligonucleotide(ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of bc1-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bc1-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bc1-2 ASODN combined with radiation could significantly inhibit expression of Bc1-2 protein in Raji cells (P<0.05). Cells treated with Bc1-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bc1-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bc1-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

  9. Effect of magnetic nanoparticles on apoptosis and cell cycle induced by wogonin in Raji cells

    Wang XM


    Full Text Available Lei Wang1,2,*, Haijun Zhang1,2,*, Baoan Chen1,2, Guohua Xia1,2, Shuai Wang1,2, Jian Cheng1,2, Zeye Shao1,2, Chong Gao1,2, Wen Bao1,2, Liang Tian1,2, Yanyan Ren1,2, Peipei Xu1,2, Xiaohui Cai1,2, Ran Liu1,2, Xuemei Wang3 1Department of Hematology and Oncology, Zhongda Hospital, Medical School, 2Faculty of Oncology, Medical School, 3State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory, Southeast University, Nanjing, China*These authors contributed equally to this workAbstract: Traditional Chinese medicine is gradually becoming a new source of anticancer drugs. One such example is wogonin, which is cytotoxic to various cancer cell lines in vitro. However, due to its low water solubility, wogonin is restricted to clinical administration. Recently, the application of drug-coated magnetic nanoparticles (MNPs to increase water solubility of the drug and to enhance its chemotherapeutic efficiency has attracted much attention. In this study, wogonin was conjugated with the drug delivery system of MNPs by mechanical absorption polymerization to fabricate wogonin-loaded MNPs. It was demonstrated that MNPs could strengthen wogonin-induced cell inhibition, apoptosis, and cell cycle arrest in Raji cells by methylthiazol tetrazolium assay, flow cytometer assay, and nuclear 4',6-diamidino-2-phenylindole staining. Furthermore, the molecular mechanisms of these phenomena were explored by western blot, in which the protein levels of caspase 8 and caspase 3 were increased significantly while those of survivin and cyclin E were decreased significantly in wogonin-MNPs group. These findings suggest that the combination of wogonin and MNPs provides a promising strategy for lymphoma therapy.Keywords: wogonin, magnetic nanoparticles, Raji cell, apoptosis, cell cycle, caspase 8, caspase 3, survivin, cyclin E

  10. Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity.

    Townsend, Michelle; Peck, Connor; Meng, Wei; Heaton, Matthew; Robison, Richard; O'Neill, Kim


    Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30-60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Fisetin targets phosphatidylinositol-3-kinase and induces apoptosis of human B lymphoma Raji cells

    Ji Yeon Lim


    Full Text Available Aberrant regulation of phosphatidylinositol-3-kinases (PI3Ks is known to be involved in the progression of cancers. PI3K-binding flavonoids such as quercetin and myricetin have been shown to inhibit PI3K activity, but the direct targeting of fisetin to PI3K has not been established. Here, we carried out an in silico investigation of fisetin binding to PI3K and determined fisetin’s inhibitory activity in enzymatic and cell-based assays. In addition, fisetin induced apoptosis in human Burkitt’s lymphoma Raji cells by inhibiting both PI3Ks and mammalian target of rapamycin (mTOR. Our results indicate that fisetin may serve as a natural backbone for the development of novel dual inhibitors of PI3Ks and mTOR for the treatment of cancer.

  12. Nonylphenol decreases viability and arrests cell cycle via reactive oxygen species in Raji cells.

    Qi, Yongmei; Zhang, Yingmei; Liu, Yingxia; Zhang, Wenya


    4-Nonylphenol (NP), an environmental contaminant commonly found in water systems, has been documented to have adverse effects on human health. In the current study, the effects of NP on the survival, reactive oxygen species (ROS) production and cell cycle distribution of human Raji cells, a human lymphoblastoid cell line with B cell characteristics, were investigated. Furthermore, N-Acetyl-Cysteine (NAC) was used to explore the underlying mechanisms. The results showed that NP dramatically reduced cell viability along with the induction of ROS in a dose dependent manner, and cell survival was recovered by NAC pretreatment. Most strikingly, NP exposure altered the cell cycle profile, mainly leading to the accumulation of cells in the G2/M phase. Pretreatment of Raji cells with NAC attenuated the NP-induced G2/M cell cycle arrest. Taken together, the results suggest NP exhibits cytotoxic effects on Raji cells by decreasing cell viability and inducing G2/M cell cycle arrest, in a ROS dependent manner. Copyright © 2011 Elsevier GmbH. All rights reserved.

  13. Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

    ZHAO Ya Ning; LI Qiang


    Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

  14. Adenovirus mediated BIMS transfer induces growth supression and apoptosis in Raji lymphoma cells.

    Zhao, Ya Ning; Li, Qiang


    To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biological processes of BIM activation so as to reverse the chemoresistance of cancer cells. BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus (Ad-BIMS) was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTT and flow cytometry. These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  15. Sodium butyrate-induced death-associated protein kinase expression promote Raji cell morphological change and apoptosis by reducing FAK protein levels

    Hai-tao ZHANG; Zhe-ling FENG; Jun WU; Ya-jun WANG; Xia GUO; Nian-ci LIANG; Zhen-yu ZHU; Jian-quan MA


    Aim:To investigate the role of death-associated protein kinase (DAPK) on the apoptosis of Raji cells induced by sodium butyrate. Methods:The apoptosis of Raji cells were induced by sodium butyrate for 2,4,6,8,and 10 d. Simultaneity,the Raji cells were inhibited to adhere on culture flask by polyHEME. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and the cell apoptosis percentage was estimated by flow cytometry. DAPK and focal adhesion kinase (FAK) expression were measured by Western blotting.Coding sequence on the C-terminal of DAPK,which can suppress the function of DAPK,was tranfected into the Raji cells to investigate whether the C-terminal of DAPK could inhibit the apoptosis of Raji cells induced by sodium butyrate. Results:After being treated with sodium butyrate,the Raji cells expressed DAPK and displayed many protrusions to adhere onto the culture flask. The Raji cells were susceptive to apoptosis when they were inhibited adhesion by polyHEME. At that time,the cell viability decreased,the cell apoptosis percentage increased and the protein levels of total FAK were reduced. The Raji cells,which were transfected with the coding region on the C-terminal of DAPK,sustained apoptosis and the FAK protein level when treated with sodium butyrate. Conclusion:Sodium butyrate induced DAPK expression. It caused the Raji cells to display many protrusions all around the cells and adhere onto the culture flask. DAPK expression prompted apoptosis by reducing the FAK protein level in sodium butyrate induced Raji cells.

  16. Study on the apoptosis of Raji cell line induced by arsenic trioxide and its correlation with Survivin gene%三氧化二砷诱导Raji细胞凋亡与Survivin基因关系的研究

    Yi Long; Huimin Li; Chen Qing; Hua Liu; Yanli Zhang; Meijia Yu


    Objective:To investigate the apoptosis induction by arsenic trioxide (As2O3) in Raji cells and its correlation with cell cycle arrest and expression of the Survivin gene.Methods:After Raji cells were treated with As2O3 in different concentrations (1,2,4 and 8 μM),for 24,48 and 72 h,respectively,and cell proliferation was tested by MTT assay.Apoptosis was observed with electron microscope end DNA electrophoresis.The distribution of cell cycles and cell apoptosis were detected by flow cytometry.Expression of the Survivin gene was determined by real-time quantitative RT-PCR.Results:As2O3 (1-8 μM) inhibited Raji cells growth effectively in a dose- and time-dependent manner.As2O3 at 2-8μM could induce cell apoptosis and cell cycle arrest.However,As2O3 (1 μM) inhibited Raji proliferation only by cell cycle arrest,without any symptoms of cell apoptosis.At the same time,Survivin gene expression was down-regulated after the treatment.Conclusion:As2O3 could induce substantial proliferation inhibition,cell cycle arrest and apoptosis in Raji cell.Cell cycle arrest might be a reason why apoptosis occurs.As2O3 can markedly down-regulate expression of the Survivin gene in a dose- and timedependent manner.The down-regulated Survivin gene might be leading to cell apoptosis by As2O3.

  17. Influence of Decitabine on Demethylation of P15INK4B Gene and the Growth and Apoptosis of Burkitt Lymphoma Raji Cells

    LIU Qiao


    Full Text Available Objective: To explore the methylation status of P15INK4B gene and the biochemical influence of decitabine on the demethylation of P15INK4B gene and the growth and apoptosis of Burkitt lymphoma Raji cells. Methods: Trypan blue was used to test the effects of different concentrations of decitabine on cell growth curve of Burkitt lymphoma Rajj cells. Cell apoptostic rate was detected by flow cytometry (FCM. The expression of P15INK4B gene was detected by reverse transcription-polymerase chain reaction (RT-PCR and the degree of methylation of P15INK4B gene by methylation-specific PCR (MSP. Results: Different concentrations of decitabine had an inhibiting effect on the proliferation of Raji cells, and promote the apoptosis of Raji cells. After 48-h treatment of decitabine, the mRNA expression of P15INK4B gene of Raji cells was up-regulated in a dose-dependent manner by inducing the demethylation of P15INK4B gene. . Conclusion: There exists hypermethylated P15INK4B gene in Burkitt lymphoma Raji cells which makes P15INK4B gene down-regulated. However, decitabine can up-regulate the mRNA expression of P15INK4B gene through inducing the demethylation of P15INK4B gene, thus inhibiting the proliferation of lymphoma Raji cells.

  18. Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells

    García-Pérez Blanca Estela


    Full Text Available Abstract Background The classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB, Mycobacterium smegmatis (MSM, and Salmonella typhimurium (ST. Results Our observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes, and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection. Conclusions Raji B cells can internalise S. typhimurium and mycobacteria through an active process, such as

  19. The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells.

    Henrich, Silke; Cordwell, Stuart J; Crossett, Ben; Baker, Mark S; Christopherson, Richard I


    Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular 'housekeeping' metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt's lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% (n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% (n=90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.

  20. Identification of Significant Pathways Induced by PAX5 Haploinsufficiency Based on Protein-Protein Interaction Networks and Cluster Analysis in Raji Cell Line

    Jia Gu


    Full Text Available PAX5 encodes a transcription factor essential for B-cell differentiation, and PAX5 haploinsufficiency is involved in tumorigenesis. There were few studies on how PAX5 haploinsufficiency regulated genes expression to promote tumorigenesis. In this study, we constructed the cell model of PAX5 haploinsufficiency using gene editing technology in Raji cells, detected differentially expressed genes in PAX5 haploinsufficiency Raji cells, and used protein-protein interaction networks and cluster analysis to comprehensively investigate the cellular pathways involved in PAX5 haploinsufficiency. The clusters of gene transcription, inflammatory and immune response, and cancer pathways were identified as three important pathways associated with PAX5 haploinsufficiency in Raji cells. These changes hinted that the mechanism of PAX5 haploinsufficiency promoting tumorigenesis may be related to genomic instability, immune tolerance, and tumor pathways.

  1. Identification of Significant Pathways Induced by PAX5 Haploinsufficiency Based on Protein-Protein Interaction Networks and Cluster Analysis in Raji Cell Line

    Gu, Jia; Li, TongJuan; Zhao, Lei; Liang, Xue; Fu, Xing; Wang, Jue; Shang, Zhen; Zhou, Jianfeng


    PAX5 encodes a transcription factor essential for B-cell differentiation, and PAX5 haploinsufficiency is involved in tumorigenesis. There were few studies on how PAX5 haploinsufficiency regulated genes expression to promote tumorigenesis. In this study, we constructed the cell model of PAX5 haploinsufficiency using gene editing technology in Raji cells, detected differentially expressed genes in PAX5 haploinsufficiency Raji cells, and used protein-protein interaction networks and cluster analysis to comprehensively investigate the cellular pathways involved in PAX5 haploinsufficiency. The clusters of gene transcription, inflammatory and immune response, and cancer pathways were identified as three important pathways associated with PAX5 haploinsufficiency in Raji cells. These changes hinted that the mechanism of PAX5 haploinsufficiency promoting tumorigenesis may be related to genomic instability, immune tolerance, and tumor pathways. PMID:28316978

  2. A Metabonomic Study of the Effect of Methanol Extract of Ginger on Raji Cells Using 1HNMR Spectroscopy

    N. Parvizzadeh


    Full Text Available Cancer is currently a major international health problem. The development of resistance to chemotherapy has resulted in the search for herbal drugs. Ginger is a medicinal plant with several clinical applications. Metabolomics is a simultaneous detection of all the metabolites by use of 1HNMR or mass spectroscopy and interpretation by modeling software. The purpose of this study was to detect the altered metabolites of Raji cells in the presence of ginger extract in vitro. Cells were cultured in the presence and absence of methanolic ginger extract in RPMI medium. IC50 determined by MTT and lipophilic and hydrophilic extracts were prepared from control and treated groups which were analyzed by 1HNMR. The IC50 was 1000 μg/mL. Modeling of spectra was carried out on the two groups using OSC-PLS with MATLAB software and the main metabolites detected. Further analysis was carried out using MetaboAnalyst database. The main metabolic pathways affected by the ginger extract were detected. Ginger extract was seen to effect the protein biosynthesis, amino acid, and carbohydrate metabolism and had a strong cytotoxic effect on Raji cells in vitro.

  3. Differentially expressed nuclear proteins in human CCRF-CEM, HL-60, MEC-1 and Raji cells correlate with cellular properties.

    Henrich, Silke; Crossett, Ben; Christopherson, Richard I


    The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.

  4. Shiga Toxin 1, as DNA Repair Inhibitor, Synergistically Potentiates the Activity of the Anticancer Drug, Mafosfamide, on Raji Cells

    Piero Sestili


    Full Text Available Shiga toxin 1 (Stx1, produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77, causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05–0.1 pM, inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.

  5. Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

    Di Renzo Livia


    Full Text Available Abstract Background EBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ, sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitt's lymphoma (BL cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents. Results Our data show that, following EBV activation, the latent membrane protein 1 (LMP1 and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression. Conclusion These findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells.

  6. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Safar Farajnia


    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  7. CD20 monoclonal antibody targeted nanoscale drug delivery system for doxorubicin chemotherapy: an in vitro study of cell lysis of CD20-positive Raji cells.

    Jiang, Shuang; Wang, Xiaobo; Zhang, Zhiran; Sun, Lan; Pu, Yunzhu; Yao, Hongjuan; Li, Jingcao; Liu, Yan; Zhang, Yingge; Zhang, Weijing

    A monoclonal antibody targeted nanoscale drug delivery system (NDDS) for chemotherapy was evaluated in CD20-positive Raji cells in vitro. Nanoparticles were formed by the assembly of an amphiphilic polymer consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethyleneglycol-2000 (DSPE-PEG2000). Active carbon nanoparticles (ACNP) were conjugated to the chemotherapeutic agent, doxorubicin (DOX), and the nanoliposome carrier, DSPE-PEG2000 and DSPE-PEG2000-NH2 conjugated to the human anti-CD20 monoclonal antibody that targets B-lymphocytes. This monoclonal antibody targeted nanoparticle delivery system for chemotherapy formed the active NDDS complex, ACNP-DOX-DSPE-PEG2000-anti-CD20. This active NDDS was spherical in morphology and had good dispersion in the culture medium. When compared with the effects on CD20-negative YTS cells derived from natural killer/T-cell lymphoma, the active NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20, demonstrated DOX delivery to CD20-positive Raji cells derived from Burkitt's lymphoma (B cell lymphoma), resulting in increased cell killing in vitro. The intracellular targeting efficiency of the ACNP-DOX-DSPE-PEG2000-anti-CD20 complex was assessed by confocal laser microscopy and flow cytometry. The findings of this in vitro study have shown that the DSPE-PEG2000 polymeric liposome is an effective nanocarrier of both a monoclonal antibody and a chemotherapy agent and can be used to target chemotherapy to specific cells, in this case to CD20-positive B-cells. Future developments in this form of targeted therapy will depend on the development of monoclonal antibodies that are specific for malignant cells, including antibodies that can distinguish between lymphoma cells and normal lymphocyte subsets.

  8. 苦参碱对Raji细胞凋亡的影响及其机制%Effect and Mechanism of Matrine on Apoptosis of Raji Cells

    刘占术; 罗章琴; 谢波; 薛乾富; 肖悦; 杨泽松; 陈建斌


    Objective: To invest the effect and mechanism of matrine on apoptosis of human Burkitt's lymphoma Raji cells. Methods:Raji cells were cultured in vitro and treated by different final concentrations (0. 4,0. 8,1.6 mg/mL) of matrine or combined with SB203580( p38 MAPK inhibitor) before matrine was added, then cocultured for 48 h, cell apoptosis rate was detected by Annexin V-FITC/PI double staining method and the P-p38 MAPK, Fas, FasL protein expresssion of Raji cells were evaluated by Western blot. Results: After cells were treated by matrine ( O. 4,0. 8,1.6 mg/mL), the corresponding total apoptosis rate ( 15. 77 ± O. 53 ) %,(27. 88 ±1. 52)%, (48. 08 ±2. 87)% ,had statistical significance compared with SB203580 groups ( 11.48 ±0. 64)%, ( 19. 34 ±O. 91 ) %, (33.98 ± 1.26) % (P < 0. 05 or P < 0. 01 ), and control group ( 8. 78 ± 0. 66) % ( P < O. 05 or P < O. 01 ). As the concentration of matrine gradually increased, the protein expresssion levels of P-p38MAPK, Fas, FasL increased, and decreased after SB203580 were added, the correlation of P-p38MAPK and Fas, FasL was obvious. Conclusion: Matrine can upregulation of Fas and FasL to promote the apoptosis of Raji cells ,it may be related to p38MAPK Activation.%目的:探讨活化的038MAPK在苦参碱(Matrine,Mat)诱导人Burkitt's淋巴瘤Raji细胞凋亡过程中对Fas/FasL的影响.方法:选用Raji细胞进行体外培养,用苦参碱(终浓度为0.4、0.8、1.6 mg/mL)和在此基础上加入SB203580(p38 MAPK抑制剂)作用48 h后,分别采用Annexin V-FITC/PI双染法检测细胞凋亡率和Western blot检测P-p38MAPK、Fas、FasL蛋白表达的变化.结果:终浓度为0.4、0.8、1.6 mg/mL苦参碱对Raji细胞总凋亡率显著高于SB203580组及对照组(P<0.05或P<0.01);Western blot显示P-p38MAPK、Fas、FasL蛋白表达水平随苦参碱浓度的提高而增加,并且在加入SB203580后降低.P-p38MAPK与Fas、FasL具有明显相关性.结论:苦参碱可能通过活化p38

  9. Affinity Capillary Electrophoresis:Study of the Binding of HIV-1 gp41 with a Membrane Protein (P45) on the Human B Cell Line,Raji


    Affinity capillary electrophoresis has been used to study the interaction between a membrane protein (P45) isolated from the Human B cell line, Raji, and rsgp41. P45, rsgp41 and the complexes were well resolved. The entire separation was achieved in less than 3min. Formations of two kinds of stable P45-rsgp41 complexes were confirmed based on migration time comparison; the binding equilibrium was achieved as soon as two proteins were mixed. The results indicate that the interaction between P45 and rsgp41 is strong with a fast association rate and a slow dissociation rate, and there are at least two kinds of binding sites with different binding constants between P45 and rsgp41.

  10. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.


    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

  11. Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells

    SUN Chunyan; LIU Xinyue; CHEN Yan; LIU Fang


    The anticancer activity of trichostain A (TSA) on human B cell non-Hodgkin's lymphoma and its mechanism were explored. The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells (NPBMNC) was studied by MTT assay. The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling (TUNEL). The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time- and concentration-dependent manner.Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.

  12. Cell viability assays: introduction.

    Stoddart, Martin J


    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  13. B cell helper assays.

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo


    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  14. Anticancer Effect of Curcumin on B Cell non- Hodgkin's Lymphoma

    SUN Chunyan; LIU Xinyue; CHEN Yan; LIU Fang


    To explore the anticancer effect of curcumin on human B cell non-Hodgkin's lymphoma and compare its effects on human B cell non-Hodgkin's lymphoma cells and normal peripheral blood mononuclear cells (NPBMNCs). MTT assay was used to study the effect of curcumin on the growth of Raji cells and NPBMNCs. The effect of curcumin on the apoptosis of Raji cells and NPBMNC were studied by flow cytometry and TDT-mediated dUTP nick and labeling (TUNEL). The effect of curcumin on the cell cycle of Raji cells were examined by propidium iodide staining flow cytometry. The results showed that curcumin strongly inhibited ±1.82 μmol/L and curcumin induced Raji cell apoptosis in a time- and dose-dependent manner. Raji cells treated with curcumin showed curcumin did not demonstrate apparent proliferation inhibition and apoptosis induction in NPBMNCs. It was concluded that curcumin is able to inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Morever, curcumin has low toxicity on NPBMNCs but can selectively induce apoptosis in Raji cells.

  15. Inspective investigation on Atlantic pomfret (Brama raji)

    Antonio Panebianco; Piergigi Ferrara; Daniele Muscolino; Filippo Giarratana


    The present study was carried out on 21 specimens of Brama raji collected at fish markets of Sicily and Calabria between april 2011 and march 2012. The detection of total Enterobacteriaceae, Vibrio spp. and Specific Spoilage Bacteria from gills, intestine, skin and muscle during storage (336h) was carried out. 34 Vibrio spp. (rpoA+) strains were isolated from gills and skin. The subsequent identification of species by multiplex PCR (gene collagenase) allowed to establish that 33 strains were ...

  16. Lidamycin Induces Apoptosis of B-Cell Lymphoma Cells and Inhibits Xenograft Growth in Nude Mice

    Hong Fang; Shenghua Zhang; Qingfang Miao; Dongsheng Xiong; Yongsu Zhen


    OBJECTIVE To study the cytotoxicity of Lidamycin (LDM) and its induction of apoptosis in Raji and Daudi cells of B-cell lymphoma, and the inhibition of growth of the lymphoma Raji xenograft in nude mice.METHODS MTT assay was used to observe the inhibition by LDM on the proliferation of the Raji and Daudi cells. Annexin V-FITC/PI double-stain, in combination with flow cytometry (FCM), was used to determine the induction of apoptosis by LDM in Raji cells. The B-cell lymphoma Raji xenograft model in nude mice was set up to detect the in vivo antitumor activity of LDM.RESULTS LDM markedly inhibited the proliferation of the Raji and Daudi cells in vitro, with IC50 values of 7.13×10-11 mol/L and 2.91×10-10 mol/L, respectively. The apoptotic rates of Raji cells were respectively 77.98% and 67.63% at 0.5 nmol/L and 0.25 nmol/L of LDM, indicating an obvious induction of apoptosis in Raji cells. LDM inhibited the formation and growth of human B-cell lymphoma Raji xenograft in nude mice. The inhibition rates of tumor growth were respectively 74.9% and 65.2% in LDM at dosage group of 0.05 mg/kg and 0.025 mg/kg, suggesting an apparent prolongation of survival time in the nude mouse bearing lymphoma.CONCLUSION LDM can effectively induce apoptosis of the B-cell lymphoma cells and inhibit the xenograft growth in nude mice.

  17. Raji cell apoptosis-inducing effects of matrine and its effect on Fas/FasL protein expression%苦参碱对Raji细胞凋亡及凋亡相关蛋白Fas/FasL表达的影响

    刘占术; 陈建斌; 汤为学; 杨泽松; 黄曦; 黄宗干


    AIM: To invest the role of matrine-induced apoptosis of Human Burkitt's lymphoma Raji cells and its mechanism. METHODS: The proliferation inhibition rate of Raji cells was performed by MTI' analysis. Cell apoptosis rate was detected by AnnexinV-FITC/PI double staining method. The Fas, FasL and Caspase-3 protein expresssion of Raji cells treated with matrine were evaluated by Western blot. RESULTS: After Raji cells were cocuhured with matrine 0. 2 - 1.6 g/L for 24,48,72 h in vitro, the cell proliferation was inhibited in time-and dosedependent manner, its half maximal inhibitory concentration( IC_(50) )of 48 h was 1.34 g/L. When the Raji cells were cultured for 48 h by matrine 0.4,0. 8,1.6 g/L, the total apoptosis rate 15.10%, 27.88% ,48.08% detected by AnnexinV-FITC/PI double staining, had statistical significance compared with control group 8.78%, ( P < 0.05 or P < 0. 01 ). The protein expresssion levels of Fas, Fasl and Caspase3 increased after Raji cells were treated by matrine 0.4,0. 8,1.6 g/L with a concentration-effect relationship. CONCLUSION : Matrine can induce the apoptosis of Raji cells in vitro, one of its possible mechanisms is that matrine activates caspase-3 through upregulafion of Fas and FasL.%目的:探讨中药苦参碱对人Burkitt's淋巴瘤Raji细胞凋亡的作用及其可能机制.方法:应用MTT法测定苦参碱对Raji细胞生长的抑制作用;AnnexinV-FITC/PI双染法检测细胞凋亡率;Western Blot检测Fas,FasL,caspase-3蛋白的表达量.结果:苦参碱0.2~1.6 g/L作用Raji细胞24,48,72 h后,对Raji细胞生长具有增殖抑制作用,且呈明显的量效和时效关系,48 h半数抑制浓度(IC_(50))为1.34 s/L.苦参碱0.4,0.8,1.6 s/L组作用48 h后,Raji细胞总凋亡率分别为15.10%,27.88%,48.08%,与对照组8.78%相比较差异具有统计学意义(P<0.05,P<0.01).Western Blot检测结果显示Fas,FasL,caspase-3蛋白表达水平随苦参碱浓度的提高而增加.结论:一定浓度苦参碱可诱导Raji细胞发生

  18. Inhibition of proliferation of human non-Hodgkin's lymphoma Raji cells by small interference RNA silencing Pokemon gene%siRNA 沉默 Pokemon 基因抑制人非霍奇金淋巴瘤 Raji细胞的增殖

    董珂; 蒲业迪; 刘琼; 代广霞; 李丽珍; 李颢; 王玲玲; 王鲁群


    Objective Small interference RNA ( siRNA) was used to silence Pokemon gene in human non-Hodgkin's lymphoma Raji cells, then to observe the change of proliferation of Raji cells and explore its possible molecular mecha-nisms.Methods Pokemon-targeted siRNA was constructed with lentivirus vector and transfected to Raji cells. RT-PCR and Western blotting were adopted to confirm the silence effect of Pokemon gene in Raji cells, which were then used to determine the mRNA and protein expressions of bcl-6 and mutant p53.Flow cytometry was used to detect the cell apoptosis of each group.Results Pokemon-siRNA constructed with lentivirus vector could efficiently silence the expression of Pokemon gene in Raji cell (P<0.05).After that, the mRNA and protein expressions of bcl-6 and mutant p53 were significantly decreased (P<0.05) and the cell apoptosis rate was markedly elevated compared with the controls.Conclusion The siRNA Pokemon gene silencing promotes human non-Hodgkin's lymphoma Raji cells apoptosis by lowering the expressions of bcl-6 and mutant p53 gene and protein and inhibiting the proliferation.Poke-mon gene is expected to become a new target for non-Hodgkin's lymphoma treatment.%目的:采用siRNA干扰技术沉默人非霍奇金淋巴瘤Raji细胞中Pokemon基因,观察Raji细胞增殖活性的变化,并探讨其可能的分子机制。方法构建靶向Pokemon基因的siRNA重组慢病毒载体并转染Raji细胞。采用实时定量PCR法和Western blotting法检测Raji细胞Pokemon基因的沉默效果,在Raji细胞中沉默Pokemon基因的表达后采用实时定量PCR法和Western blotting法检测bcl-6和突变型p53表达水平的变化;采用流式细胞术检测Pokemon基因沉默后Raji细胞凋亡的情况。结果利用Pokemon靶向siRNA重组慢病毒载体感染Raji细胞有效沉默Raji细胞Pokemon基因表达(P<0.05)。沉默Pokemon基因后,bcl-6和突变型p53基因和蛋白表达均显著降低(P<0.05)

  19. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose


    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raj...

  20. Cytotoxic assay of endophytic fungus 1.2.11 secondary metabolites from Brucea javanica (L Merr towards cancer cell in vitro

    Pratiwi Sudarmono


    Full Text Available Cytotoxic assay of secondary metabolite endophytic fungus 1.2.11 from Brucea javanica (L Merr has been carried out. Brucea javanica fruit collected from Cianjur was used in this experiment. Cytotoxic assay was done on Raji, NS-1, HeLa and Vero cells. The observation was done for 24 hours and also for 48 hours. IC50 was calculated using the Rich and Muench theory. To observe the working mechanism of cytotoxic process, DNA staining with etidium bromide and acridine orange was conducted. The cytotoxic assay of endophytic fungi 1.2.11 showed an IC50 of 58.35 μg/ml, 88.39 μg/ml on Raji cell,; 162.09 μg/ml, 66.24 μg/ml on NS cell; 361.21 μg/ml, 219.97 μg/ml on HeLa cell; and lastly 1075.18 μg/ml, 656.82 μg/ml on Vero cell after 24 and 48 hour incubation respectively. The results of this study showed that secondary metabolite of endophytic fungus 1.2.11 has selective cytotoxic effect towards cancer cell and also showed that it might cause apoptosis in NS-1cell. (Med J Indones 2006; 15:137-44 Keywords: Brucea javanica (L. Merr, endophytic microbe, Cytotoxic assay, endophytic isolate 1.2.11, apoptosis

  1. Curcumin-induced Histone Acetylation in Malignant Hematologic Cells

    Junbin HU; Yan WANG; Yan CHEN


    This study investigated the inhibitory effects of curcumin on proliferation of hemato-logical malignant cells in vitro and the anti-tumor mechanism at histone acetylation/histone deacety-lation levels.The effects of curcumin and histone deacetylase inhibitor trichostatin A (TSA) on the growth of Raji cells were tested by MTT assay.The expression of acetylated histone-3 (H3) in Raji,HL60 and K562 cells,and peripheral blood mononuclear cells (PBMCs) treated with curcumin or TSA was detected by immunohistochemistry and FACS.The results showed curcumin inhibited pro-liferation of Raji cells significantly in a time- and dose-dependent fashion,while exhibited low toxic-ity in PBMCs.Curcumin induced up-regulation of the expression of acetylated H3 dose-dependently in all malignant cell lines tested.In conclusion,curcumin inhibited proliferation of Raji cells selec-tively,enhanced the level of acetylated H3 in Raji,HL60,and K562 cells,which acted as a histone deacetylase inhibitor like TSA.Furthermore,up-regulation of H3 acetylation may play an important role in regulating the proliferation of Raji cells.

  2. Inspective investigation on Atlantic pomfret (Brama raji

    Antonio Panebianco


    Full Text Available The present study was carried out on 21 specimens of Brama raji collected at fish markets of Sicily and Calabria between april 2011 and march 2012. The detection of total Enterobacteriaceae, Vibrio spp. and Specific Spoilage Bacteria from gills, intestine, skin and muscle during storage (336h was carried out. 34 Vibrio spp. (rpoA+ strains were isolated from gills and skin. The subsequent identification of species by multiplex PCR (gene collagenase allowed to establish that 33 strains were V. alginolyticus and one was V. parahaemolyticus. The anatomopathological examination of muscle tissue showed that 15 specimens (71.x4% were positive for the presence of larvae of Gymnorhynchus gigas. The determination of TVB-N and TMA-N was made on muscle portions around the parasite, on larvae of G. gigas and on muscle portions parasite-free. The TVB-N and TMA-N contents of each sample were measured using the Conway microdiffusion method. The higher values of TVB-N (31.6 mg/100g and TMA-N (8.5 mg/100g were observed at 336 hours of storage. No statistically significant differences between muscle parasite-free and muscle around the parasite for TVB-N and TMA-N were observed during storage. Parasites of genus Koellikeria filicollis, Sphiriocephalus tergestinus and Anisakis spp. were also observed.

  3. Bioluminescence assay for cell viability.

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N


    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  4. 电磁辐射对Raji细胞损伤效应及其Bax和Bcl-2蛋白表达的影响%Electromagnetic Radiation Induced Damage in Raji Cells and Effects on Bax and Bcl-2 Protein Expression

    王炜; 崔华娟; 王德文; 左红艳; 彭瑞云; 王晓民; 姚华


    目的 比对性研究电磁脉冲(electromagnetic pulse,EMP)、S波段高功率微波(S-band high power microwave,S-HPM)和X波段高功率微波(X-band high power microwave,X-HPM)三种不同波段电磁辐射,对Raji细胞的损伤效应和对其Pax和Bcl-2蛋白表达的影响,探讨其损伤效应的相关分子机制.方法常规培养Raji细胞,以EMP、S-HPM和X-HPM三种不同波段的电磁波为辐照源,分别照射Raji细胞,设伪照射为对照组.于照后6h后收集细胞,用电镜观察Raji细胞超微结构的改变,并采用细胞计数的方法对损伤细胞进行半定量计数;采用敏感的蛋白质印迹(Western blot)技术检测各组Raji细胞内Bax、Bcl-2的蛋白表达,用多功能真彩色病理图像分析系统(turecolor medical image processing and analysis,CMIAS)对阳性信号条带进行定量分析.所得数据分别采用SPSS 13.0统计学软件的x2检验和一元方差分析进行统计学处理.结果 三种不同波段的电磁辐射均可导致Raji细胞超微结构发生不同程度的损伤并见凋亡小体形成,损伤程度呈X-HPM组>EMP组>S-HPM组的规律特点;三种不同波段的电磁辐射还可致Raji细胞内Pax、Bcl-2蛋白表达发生改变.与对照组比较,Bax、Bcl-2蛋白表达在EMP组和X-HPM组均明显上调(P<0.01或0.05),S-HPM组略有下调,但无统计学意义(P>0.05).各实验组间比较,S-HPM组Bax蛋白表达明显低于EMP组(P<0.01)和X-HPM组(P<0.05),S-HPM组Bcl-2蛋白表达明显高于EMP组和X-HPM组(P<0.01).两种蛋白上调或下调的程度呈现X-HPM组>EMP组>S-HPM组的规律特点.结论 电磁辐射可能通过上调Bax蛋白的表达和下调Bcl-2蛋白的表达,从而诱导Raji细胞的损伤和凋亡.%Objective To compare the damage on Raji cells and effects on Bax and Bcl-2 protein expressions induced by electromagnetic radiation of electromagnetic pulse (EMP), S-band high power microwave (S-HPM) and X-band high power microwave (X-HPM), and

  5. Comet assay on tetraploid yeast cells

    Rank, Jette; Syberg, Kristian; Jensen, Klara


    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  6. Assay of mast cell mediators

    Rådinger, Madeleine; Jensen, Bettina M; Swindle, Emily


    Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes...... regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well...

  7. Comet assay on mice testicular cells

    Sharma, Anoop Kumar


    for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity...

  8. Comet assay on mice testicular cells

    Anoop Kumar Sharma


    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  9. In vitro cytotoxic and apoptotic activity of four Persian medicine plants on human leukemia and lymphoma cells

    Fatemeh Zare Shahneh


    Full Text Available Objective: To investigate the cytotoxic and apoptotic activity of Ferulago angulata, Echinophora platyloba, Salvia officinalis and Chelidonium majus on leukemia and lymphoma cell lines, nonHodgkin ’s B-cell lymphoma (Raji, human leukemic monocyte lymphoma (U937, human acute myelocytic leukemia (KG-1A cell lines and peripheral blood mononuclear cells. Methods: C ytotoxicity was determined by the 3 - (4, 5 -dimethylthiazol- 2-yl - 2, 5-diphenyltetrazolium bromide assay. Cell viability assay was done using trypan blue exclusion experiments and cell death was identified as apoptosis using death detection ELISA. Results: Our results demonstrated that the extracts dose and time dependently suppressed the proliferation of three leukemia and lymphoma tumor cell lines (KG-1A, U937 and Raji with ascending order of IC50 values, while peripheral blood mononuclear cells were not significantly affected. Nucleosome productions in apoptotic KG-1A, U937, and Raji cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of the extracts. Conclusions: The extracts were found to time- and dose-dependently inhibit the proliferation of KG-1A, U937, and Raji cells possibly via an apoptosis-dependent pathway.

  10. Cell Culture Assay for Human Noroviruses [response

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.


    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  11. Cell based assay for hypoglycemic drugs screening

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  12. Changes in Ca2+ concentration and caspase-3 expression and their relationship in Raji cells exposed to electromagnetic radiation%电磁辐射致Raji细胞内Ca2+浓度和Caspase-3蛋白表达的改变及其相互关系

    王炜; 刘换新; 王德文; 左红艳; 彭瑞云


    目的 研究电磁脉冲(electromagnetic pulse,EMP)、S波段高功率微波(S-band high power microwave,S-HPM)、X波段高功率微波(X-band high power microwave,X-HPM)3种不同波段电磁辐射对Raji细胞内Ca2+浓度、Caspase-3蛋白表达的影响及两者之间的相互关系,探讨电磁辐射损伤的相关调控机制.方法 常规培养Raji细胞,用S-HPM、X-HPM和EMP 3种不同波段的电磁辐射照射Raji细胞,设假照射为对照组.于对数生长期照射,照射后6h收集细胞,采用激光扫描共聚焦显微镜检测细胞内Ca2+浓度的改变,采用免疫印迹法(Western blot)检测细胞内Caspase-3蛋白的表达.结果 3种不同波段的电磁辐射照射后6h,Raji细胞内Ca2+浓度增加(EMP组、S-HPM组、X-HPM组Ca2+荧光强度分别为69.56±1.71、50.06±1.89、70.68±1.59),Caspase-3蛋白表达水平上调(EMP组、S-HPM组、X-HPM组Caspase-3蛋白表达水平分别为0.964±0.12、0.586±0.16、0.970±0.07),各辐射组Raji细胞Ca2+荧光强度、Caspase-3蛋白表达水平均明显高于对照组(分别为43.08±2.08、0.444 ±0.13),差异均有统计学意义(P<0.01);EMP组和X-HPM组Ca2+荧光强度、Caspase-3蛋白表达水平均明显高于S-HPM组,差异亦有统计学意义(P<0.01):EMP与X-HPM组间的差异无统计学意义(P>0.05),但均较S-HPM组上调明显,差异均有统计学意义(P<0.01).辐射组Ca2+浓度增加的规律和Caspase-3蛋白表达上调的规律具有一致性,线性回归分析结果显示,Caspase-3蛋白的表达随着Ca2+浓度的增高而上调,二者具有一定的相关性(P<0.01).结论 3种辐射损伤效应可能是通过增加细胞内Ca2+浓度进一步诱导Caspase-3过表达所致.%Objective To study the effects of electromagnetic pulse (EMP),S-band high power microwave (S-HPM),and X-band high power microwave (X-HPM) on the Ca2+ concentration and caspase-3 expression in Raji cells and the rclationship between Ca2+ concentration and caspase-3 expression

  13. Cryptosporidium cell culture infectivity assay design.

    King, B J; Keegan, A R; Robinson, B S; Monis, P T


    Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

  14. 21 CFR 864.7100 - Red blood cell enzyme assay.


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity...

  15. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    Buch Karl


    Full Text Available Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98 were included in the experiment to study its principal and general applicability.

  16. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  17. Optimization of cell motility evaluation in scratch assay

    Gotsulyak N. Ya.


    Full Text Available A scratch test is one of the most popular methods of classical cell migration assay in a monolayer culture. At the same time, the scratch assay has some disadvantages that can be easily corrected. Aim. Optimization the existent scratch assay on the base of detection of scratch wound surface area and the length of the field of observation which is more objective and less time consuming. Methods. Scratch assay. Results. The modification of scratch assay enables to perform measurement more accurately and rapidly. This approach is more simple and eliminates the main disadvantages of the classical method. Conclusions. The procedure of scratch wound width measurement calculated on the base of detection of cell free area and the length of the field of observation is more effective than the classical wound healing assay. It will be useful for the estimation of cell migration dynamics in monolayer culture for a wide range of live cell based experiments.

  18. Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

    Maeda, Akihisa; Takahashi, Kei; Tsuchiyama, Hiromi; Oshida, Keiyu


    The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Cell-based Assays to Identify Inhibitors of Viral Disease

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong


    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  20. Inflight Assay of Red Blood Cell Deformability

    Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.


    Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

  1. Reference cells and ploidy in the comet assay

    Gunnar eBrunborg


    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  2. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto


    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular



    Objective: To investigate the F1t3 receptor expression on the surface of malignant hematopoietic cells, the effect of TNFa and dexamethasone (DXM) on its expression and the responses of those cells to recombinant human F1t3 ligand (rhFL). Methods: Eighteen malignant hematopoietic cell lines were determined for the F1t3 receptor expression by flow cytometric analysis. The effect of rhFL on the proliferation of malignant hematopoietic cells in vitro was measured using MTT assay. Results: The expressions of F1t3 receptor on the surface of Raji, Daudi, HL-60, 8266 and XG-6 cells were detected by flow cytometric analysis. Following incubation with 20 ng/ml TNFa for 24h, the number of F1t3 receptor positive cells decreased in Raji and 8266, increased in HL-60 and XG-6, and no difference in Daudi cells. After incubation with 10-6 mol/L DXM for 24h, the number of F1t3 receptor positive cells decreased in all the 5 F1t3 receptor positive cell lines. rhFL stimulated the proliferation of HL-60 and Raji cells. Conclusion: For most of the malignant hematopoietic cells, there was neither the expression of F1t3 receptor nor the response to rhFL. DXM may be useful to reduce the effect of FL on the proliferation of some F1t3 receptor positive malignant hematopoietic cells in vitro and in vivo.

  4. Epithelial cells as alternative human biomatrices for comet assay

    Emilio eRojas


    Full Text Available The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes.Over a thirty year period, the comet assay in epithelial cells has been litlle employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  5. A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays.

    Grace Ka Yan Chan

    Full Text Available In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1 significant underestimation of compound potency and efficacy, and 2 non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.

  6. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E


    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

  7. A Cell-Based Assay to Assess Hemichannel Function

    Krishnan, Srinivasan; Fiori, Mariana C.; Cuello, Luis G.; Altenberg, Guillermo A.


    Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.

  8. Real time assays for quantifying cytotoxicity with single cell resolution.

    Sonny C Hsiao

    Full Text Available A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC, antibody dependent cellular cytotoxicity (ADCC, and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

  9. Inhibitory and Cytotoxic Activities of Salvia Officinalis L. Extract on Human Lymphoma and Leukemia Cells by Induction of Apoptosis

    Abbas Azadmehr


    Full Text Available Purpose: Salvia officinalis L., also known as Maryam Goli, is one of the native plants used to Persian medicinal herbs. Hence, the objective of this study was to examine the in vitro cytotoxic activities of a standardized crude methanol extracts prepared from Salvia officinalis L., on a non-Hodgkin’s B-cell lymphoma (Raji and human leukemic monocyte lymphoma (U937, Human acute myelocytic leukemia (KG-1A and Human Umbilical Vein Endothelial (HUVEC cell lines. Methods: The effect of methanolic extract on the inhibition of cell proliferation and cytotoxic activity was evaluated by Dye exclusion and Micro culture tetrazolium test (MTT cytotoxicity assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determined whether the mechanism involves induction of apoptosis or necrosis. Results: The present results demonstrated that methanolic extract at 50 to 800 μg/ml dose and time-dependently suppressed the proliferation of KG-1A, U937 and Raji cells by more than 80% (p800 Ag/ml. Nucleosome productions in KG-1A, Raji and U937 cells were significantly increased respectively upon the treatment of Salvia officinalis L. extract. Conclusion: The Salvia officinalis L. extract was found dose and time-dependently inhibits the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway.

  10. Keratinocyte-based cell assays: their potential pitfalls.

    Zupancic, Tina; Ozir, Mateja; Törmä, Hans; Komel, Radovan; Liovic, Mirjana


    As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening compound libraries for new or better drugs is another approach to improve existing treatments for genodermatoses. However in this study, we report of the potential pitfalls when using this type of cell lines as a "reporter" system. When cell lines with different genetic backgrounds are being used in cell-based assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of days in culture). We demonstrate how culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially also giving rise to misleading data.

  11. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.

    Cory, A H; Owen, T C; Barltrop, J A; Cory, J G


    A new tetrazolium analog of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was evaluated as a substitute for MTT in the microculture screening assay for in vitro cell growth. This new tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), in the presence of phenazine methosulfate (PMS), gave a water-soluble formazan product that had an absorbance maximum at 490-500 nm in phosphate-buffered saline. The amount of colored product formed was proportional to the number of cells and the time of incubation of the cells with MTS/PMS. MTS/PMS was reactive in all the cell lines tested which included mouse leukemia L1210 cells, mouse Ehrlich tumor cells, mouse 3T3 fibroblasts, and human colon tumor cells (HT-29). HT-29 and 3T3 fibroblasts reduced MTS/PMS more efficiently than they reduced MTT. Comparable to the amount of product formed from MTT, MTS/PMS gave excellent product formation. The IC50 value for pyrazoloimidazole obtained using MTS/PMS was 200 microM; for 5-fluoro-2'-deoxyuridine, the IC50 value was 0.9 nM. These values compared very favorably with the IC50 values obtained by direct cell counts. Further, the same IC50 values were obtained when the absorbances of the formazan product in the 96-well plates were determined after different times of incubation.

  12. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  13. Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment.

    Klaus, Joseph P; Botten, Jason


    Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

  14. Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

    Zhuan-Zi Wang; Wen-Jian Li; Hong Zhang; Jian-She Yang; Rong Qiu; Xiao Wang


    AIM: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines.METHODS: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique.RESULTS: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to y-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r= 0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant.CONCLUSION: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

  15. Expanding the available assays: adapting and validating In-Cell Westerns in microfluidic devices for cell-based assays.

    Paguirigan, Amy L; Puccinelli, John P; Su, Xiaojing; Beebe, David J


    Microfluidic methods for cellular studies can significantly reduce costs due to reduced reagent and biological specimen requirements compared with many traditional culture techniques. However, current types of readouts are limited and this lack of suitable readouts for microfluidic cultures has significantly hindered the application of microfluidics for cell-based assays. The In-Cell Western (ICW) technique uses quantitative immunocytochemistry and a laser scanner to provide an in situ measure of protein quantities in cells grown in microfluidic channels of arbitrary geometries. The use of ICWs in microfluidic channels was validated by a detailed comparison with current macroscale methods and shown to have excellent correlation. Transforming growth factor-β-induced epithelial-to-mesenchymal transition of an epithelial cell line was used as an example for further validation of the technique as a readout for soluble-factor-based assays performed in high-throughput microfluidic channels. The use of passive pumping for sample delivery and laser scanning for analysis opens the door to high-throughput quantitative microfluidic cell-based assays that integrate seamlessly with existing high-throughput infrastructure.

  16. Isolation of Treg cells and Treg cell suppression/death assay



    In vitro Treg suppression assays are performed to determine the functional effect of Treg cells on CD4 T cells. They are performed by co-culturing the responding population (Tresp) with the Treg cells or control CD4 cells (Tcon cells).

  17. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    Frazier, M.E.; Samuel, J.E.


    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  18. An assay to measure adriamycin binding in osteosarcoma cells.

    Gebhardt, M C; Kusuzaki, K; Mankin, H J; Springfield, D S


    Adjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C


    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  20. Development of a Drosophila cell-based error correction assay

    Jeffrey D. Salemi


    Full Text Available Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT interactions before anaphase onset results in chromosomal instability (CIN, which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5. Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due to the activity of kinesin-14 (Ncd when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC. Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and

  1. Development of a Drosophila cell-based error correction assay.

    Salemi, Jeffrey D; McGilvray, Philip T; Maresca, Thomas J


    Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

  2. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Marcotte, Edward M.


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential ap...

  3. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    Moshal, Karni S.; Ferri-Lagneau, Karine F.; Haider, Jamil; Pardhanani, Pooja; Leung, TinChung, E-mail: [Biomedical/Biotechnology Research Institute, North Carolina Central University, North Carolina Research Campus, Nutrition Research Center, 500 Laureate Way, Kannapolis, NC 28081 (United States)


    Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell

  4. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    Pooja Pardhanani


    Full Text Available Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299 and the mouse lung adenocarcinoma (CL13. Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to

  5. Establishment of A Lymphoma Cell Line with Stable Expression of DLC-1 by Gene Transfection%稳定表达DLC-1的淋巴瘤细胞株模型的建立

    黄波; 冯敏华; 张弢; 冷海燕; 陈字


    Objective To establish a human lymphoma cell line that stably expresses deleted in human liver cancer-1 gene(DLC-1).Methods The reconstructed plasmid,pcDNA3.1-DLC-1,was transfected to the human lymphoma cell line of Raji.Then the transfected Raji cells were selected by G418.The stable overexpression of DLC-1 was identified by Western blot assay.Results The recombined plasmid,pcDNA3.1-DLC-1,was confirmed by restriction endonuclease examination and sequencing.Western-blot showed that DLC-1 protein expression was increased in the transfected Raji cells.Conclusion A human lymphoma cell line with stable expression of DLC-1 is established.%目的 建立稳定表达肝癌缺失基因-1(DLC-1)的淋巴瘤细胞株.方法 将DLC-1真核表达质粒转染人淋巴瘤细胞株Raji,G418筛选,对获得的Raji细胞株进行Western blot鉴定.结果 酶切和测序鉴定DLC-1的cDNA片段正确插入pcDNA3.1质粒,Western blot鉴定转染后Raji细胞株DLC-1高表达.结论 成功建立了稳定高表达DLC-1的淋巴瘤细胞株.

  6. High throughput RNAi assay optimization using adherent cell cytometry

    Pradhan Leena


    Full Text Available Abstract Background siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC. Methods AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM, or non-targeting labeled siRNA, siGLO Red (5 or 50 nM using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. Results After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19. Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. Conclusion This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.

  7. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  8. Cell- and biomarker-based assays for predicting nephrotoxicity.

    Huang, Johnny X; Blaskovich, Mark A; Cooper, Matthew A


    Drug-induced nephrotoxicity contributes to the failure rate of investigational drugs during clinical trials. We are still not able to accurately predict drug-induced nephrotoxicity during early drug discovery and development. There is an urgent need for a robust screening system that can identify nephrotoxic compounds before they reach the clinic. This review discusses traditional and emerging kidney injury biomarkers that are used for the determination of nephrotoxicity and for evaluation and diagnosis of other kidney diseases. The potential for in vivo biomarkers to predict renal toxicity in high-throughput in vitro screening assays is discussed. We also compare cell types and highlight novel three-dimensional (3D) culture technologies with potential for in vitro prediction of nephrotoxicity. Traditional cell culture methods and cytotoxicity assays are well established as in vitro tests for nephrotoxicity but the correlation with in vivo results is extremely poor. Recently validated renal biomarkers show promise for early in vivo detection of nephrotoxicity, but have yet to be successfully applied for in vitro prediction of drug-induced nephrotoxicity. Advanced culture technologies 'kidney-on-a-chip' and 3D culture can produce biomarker signatures from relevant kidney cell types that show promise as better predictive systems.

  9. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    Varamini, P; Soltani, M; Ghaderi, A


    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  10. Automated cell-based assay for screening of aquaporin inhibitors.

    Mola, Maria Grazia; Nicchia, Grazia Paola; Svelto, Maria; Spray, David C; Frigeri, Antonio


    Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 microM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders.

  11. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity

    Baseler Michael


    Full Text Available Abstract Background The interferon-γ (IFN-γ ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. Methods We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY and an in vitro stimulated anti-Flu matrix peptide (FMP-specific CTL. Results When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Conclusion Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT

  12. Cell based assays for anti-Plasmodium activity evaluation.

    Mokgethi-Morule, Thabang; N'Da, David D


    Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed.

  13. Cell-based assays in GPCR drug discovery.

    Siehler, Sandra


    G protein-coupled receptors (GPCRs) transmit extracellular signals into the intracellular space, and play key roles in the physiological regulation of virtually every cell and tissue. Characteristic for the GPCR superfamily of cell surface receptors are their seven transmembrane-spanning alpha-helices, an extracellular N terminus and intracellular C-terminal tail. Besides transmission of extracellular signals, their activity is modulated by cellular signals in an auto- or transregulatory fashion. The molecular complexity of GPCRs and their regulated signaling networks triggered the interest in academic research groups to explore them further, and their drugability and role in pathophysiology triggers pharmaceutical research towards small molecular weight ligands and therapeutic antibodies. About 30% of marketed drugs target GPCRs, which underlines the importance of this target class. This review describes current and emerging cellular assays for the ligand discovery of GPCRs.

  14. Measurement of DNA damage in individual cells using the Single Cell Gel Electrophoresis (Comet) assay.

    Hartley, Janet M; Spanswick, Victoria J; Hartley, John A


    The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.

  15. Establishment of a molecular embryonic stem cell developmental toxicity assay.

    Panzica-Kelly, Julieta M; Brannen, Kimberly C; Ma, Yan; Zhang, Cindy X; Flint, Oliver P; Lehman-McKeeman, Lois D; Augustine-Rauch, Karen A


    The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

  16. High content cell-based assay for the inflammatory pathway

    Mukherjee, Abhishek; Song, Joon Myong


    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  17. Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition

    Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria


    .... Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay...

  18. Cell Image Velocimetry (CIV): boosting the automated quantification of cell migration in wound healing assays.

    Milde, Florian; Franco, Davide; Ferrari, Aldo; Kurtcuoglu, Vartan; Poulikakos, Dimos; Koumoutsakos, Petros


    Cell migration is commonly quantified by tracking the speed of the cell layer interface in wound healing assays. This quantification is often hampered by low signal to noise ratio, in particular when complex substrates are employed to emulate in vivo cell migration in geometrically complex environments. Moreover, information about the cell motion, readily available inside the migrating cell layers, is not usually harvested. We introduce Cell Image Velocimetry (CIV), a combination of cell layer segmentation and image velocimetry algorithms, to drastically enhance the quantification of cell migration by wound healing assays. The resulting software analyses the speed of the interface as well as the detailed velocity field inside the cell layers in an automated fashion. CIV is shown to be highly robust for images with low signal to noise ratio, low contrast and frame shifting and it is portable across various experimental settings. The modular design and parametrization of CIV is not restricted to wound healing assays and allows for the exploration and quantification of flow phenomena in any optical microscopy dataset. Here, we demonstrate the capabilities of CIV in wound healing assays over topographically engineered surfaces and quantify the relative merits of differently aligned gratings on cell migration.

  19. Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

    Huerta, Leonor; Lamoyi, Edmundo; Báez-Saldaña, Armida; Larralde, Carlos


    In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion. Copyright 2002 Wiley-Liss, Inc.

  20. Stem cell assays: Something old, something new, something borrowed : Something old, something new, something borrowed

    van Os, R.; Kamminga, Leonie; de Haan, G.


    Numerous assays exist that measure the function of stem cells. In this article, we review in detail the history and future of existing stem cell assays. Hematopoietic stem cells (HSCs) are historically the most well studied, but new developments in stem cell research, including the claim of stem

  1. Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays.

    Hung, Paul J; Lee, Philip J; Sabounchi, Poorya; Lin, Robert; Lee, Luke P


    We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology. (c) 2004 Wiley Periodicals, Inc.

  2. Effects of digoxin on cell cycle, apoptosis and NF-κB pathway in Burkitt's lymphoma cells and animal model.

    Wang, Ting; Xu, Peipei; Wang, Fan; Zhou, Di; Wang, Ruju; Meng, Li; Wang, Xiaohui; Zhou, Min; Chen, Bing; Ouyang, Jian


    Digoxin has potential antitumor properties. This study investigated whether digoxin suppressed Burkitt's lymphoma (BL) cells. Raji and NAMALWA cells were exposed to digoxin, followed by assay of cell viability, apoptosis and cell cycle. Western blotting was used to analyze NF-κB activity. A xenograft model was established for therapeutic efficacy evaluation. Digoxin inhibited cell growth and resulted in apoptosis and cell cycle arrest (G0/G1 for Raji cells; G2/M for NAMALWA cells). Digoxin inhibited DNA synthesis and induced morphological apoptotic characteristics. Besides, digoxin inhibited NF-κB and TNF-α-stimulated NF-κB activity, and suppressed NF-κB initiating genes (Bcl-2, Bcl-xL, cyclin D1, and c-myc), however, increased p21(cip1). Digoxin activated caspase-9/3. Furthermore, digoxin inhibited xenograft tumors growth and reduced Ki-67 and c-myc. Digoxin exerted antitumor effects on BL cells in vitro and in vivo might through regulating NF-κB and caspase pathway. These outcomes highlight the potential of digoxin as a therapeutic agent for BL.

  3. Accurate non-invasive image-based cytotoxicity assays for cultured cells

    Brouwer Jaap


    Full Text Available Abstract Background The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. Results Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. Conclusions This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

  4. Preliminary Validation of Tumor Cell Attachment Inhibition Assay for Developmental Toxicants With Mouse S180 Cells



    This study was designed to explore the possibility of using ascitic mouse sarcoma cell line(S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC5o(the concentration that reduced attachment by 50% ), of these 2 chemicals was 1.2 ×10-3 mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also testedand these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

  5. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    Ramji, Ramesh; WANG, MING; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung


    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel...

  6. Clinacanthus nutans Extracts Are Antioxidant with Antiproliferative Effect on Cultured Human Cancer Cell Lines

    Yoke Keong Yong


    Full Text Available Clinacanthus nutans Lindau leaves (CN have been used in traditional medicine but the therapeutic potential has not been explored for cancer prevention and treatment. Current study aimed to evaluate the antioxidant and antiproliferative effects of CN, extracted in chloroform, methanol, and water, on cancer cell lines. Antioxidant properties of CN were evaluated using DPPH, galvinoxyl, nitric oxide, and hydrogen peroxide based radical scavenging assays, whereas the tumoricidal effect was tested on HepG2, IMR32, NCL-H23, SNU-1, Hela, LS-174T, K562, Raji, and IMR32 cancer cells using MTT assay. Our data showed that CN in chloroform extract was a good antioxidant against DPPH and galvinoxyl radicals, but less effective in negating nitric oxide and hydrogen peroxide radicals. Chloroform extract exerted the highest antiproliferative effect on K-562 (91.28±0.03% and Raji cell lines (88.97±1.07% at 100 μg/ml and the other five cancer cell lines in a concentration-dependent manner, but not on IMR-32 cells. Fourteen known compounds were identified in chloroform extract, which was analysed by gas chromatography—mass spectra analysis. In conclusion, CN extracts possess antioxidant and antiproliferative properties against cultured cancer cell lines, suggesting an alternate adjunctive regimen for cancer prevention or treatment.

  7. Clinacanthus nutans Extracts Are Antioxidant with Antiproliferative Effect on Cultured Human Cancer Cell Lines.

    Yong, Yoke Keong; Tan, Jun Jie; Teh, Soek Sin; Mah, Siau Hui; Ee, Gwendoline Cheng Lian; Chiong, Hoe Siong; Ahmad, Zuraini


    Clinacanthus nutans Lindau leaves (CN) have been used in traditional medicine but the therapeutic potential has not been explored for cancer prevention and treatment. Current study aimed to evaluate the antioxidant and antiproliferative effects of CN, extracted in chloroform, methanol, and water, on cancer cell lines. Antioxidant properties of CN were evaluated using DPPH, galvinoxyl, nitric oxide, and hydrogen peroxide based radical scavenging assays, whereas the tumoricidal effect was tested on HepG2, IMR32, NCL-H23, SNU-1, Hela, LS-174T, K562, Raji, and IMR32 cancer cells using MTT assay. Our data showed that CN in chloroform extract was a good antioxidant against DPPH and galvinoxyl radicals, but less effective in negating nitric oxide and hydrogen peroxide radicals. Chloroform extract exerted the highest antiproliferative effect on K-562 (91.28 ± 0.03%) and Raji cell lines (88.97 ± 1.07%) at 100  μ g/ml and the other five cancer cell lines in a concentration-dependent manner, but not on IMR-32 cells. Fourteen known compounds were identified in chloroform extract, which was analysed by gas chromatography-mass spectra analysis. In conclusion, CN extracts possess antioxidant and antiproliferative properties against cultured cancer cell lines, suggesting an alternate adjunctive regimen for cancer prevention or treatment.

  8. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei


    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  9. Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

    Mertsch, Sonja; Thanos, Solon


    For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

  10. Cell-of-Origin of Cancer versus Cancer Stem Cells: Assays and Interpretations.

    Rycaj, Kiera; Tang, Dean G


    A tumor originates from a normal cell that has undergone tumorigenic transformation as a result of genetic mutations. This transformed cell is the cell-of-origin for the tumor. In contrast, an established clinical tumor is sustained by subpopulations of self-renewing cancer cells operationally called cancer stem cells (CSC) that can generate, intraclonally, both tumorigenic and nontumorigenic cells. Identifying and characterizing tumor cell-of-origin and CSCs should help elucidate tumor cell heterogeneity, which, in turn, should help understand tumor cell responses to clinical treatments, drug resistance, tumor relapse, and metastatic spread. Both tumor transplantation and lineage-tracing assays have been helpful in characterizing these cancer cell populations, although each system has its strengths and caveats. In this article, we briefly review and summarize advantages and limitations of both assays in support of a combinatorial approach to accurately define the roles of both cancer-initiating and cancer-propagating cells. As an aside, we also wish to clarify the definitions of cancer cell-of-origin and CSCs, which are often interchangeably used by mistake.

  11. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Hart, Traver; Zhao, Alice; Garg, Ankit; Bolusani, Swetha; Marcotte, Edward M


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFkappaB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon), and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  12. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  13. [Cytomegalovirus isolation by conventional cell culture and shell vial assay].

    Galiano, M C; Videla, C M; Sánchez Puch, S; Carballal, G


    In immunocompromised patients, diagnosis of Cytomegalovirus (CMV) active infection is of utmost importance for the initiation, monitoring and ending of antiviral therapy. Therefore, the presence of viral replication should be demonstrated. Isolation in tissue culture is one of the standard methods. The objective of the present paper was to compare two isolation procedures for CMV: conventional cell culture (CC) and rapid shell vial (SV) assay in human fibroblasts. A total of 584 clinical samples were studied between 1991 and 1998. CMV was isolated in 14.4% of the samples, 11.8% of which were positive by SV and 7.7% by CC. Out of 84 positive samples, concordance between both methods was observed in 36% of the cases. We found that 46% of the samples were positive only by SV, while 18% were positive only by CC. The average time required for obtaining the results by CC was 22.6 +/- 2.3 days. Out of the 69 samples positive by SV, 43% were already positive after 24 hours and the rest after 48 hours. These results indicate that SV was more sensitive and rapid than CC. The main advantage of CC, despite its time-consuming process, is the ability to recover the viral strain for both antiviral susceptibility phenotypical tests and strain characterization. Furthermore, in this study, absence of CC would have resulted in the loss of 18% of the positive diagnoses. In conclusion, simultaneous use of both methods is suggested in order to obtain a rapid result and the highest sensitivity.

  14. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick


    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  15. Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum.

    Kumar, Sunil; Kanwar, Sarika; Bansal, Vivek; Sehgal, Rakesh


    Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.

  16. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Valverde Mahara


    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  17. The application of single cell gel electrophoresis or comet assay to human monitoring studies


    Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a g...

  18. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M


    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  19. Crossing barriers: the new dimension of 2D cell migration assays.

    Horssen, R. van; Hagen, T.L.M. ten


    In our body cells move in three dimensions, embedded in an extracellular matrix that varies in composition, density and stiffness, and this movement is fundamental to life. Next to 3D cell migration assays, representing these physiological circumstances, still we need 2D migrations assays to perform

  20. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W


    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

  1. A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis.

    Di Costanzo, Ezio; Ingangi, Vincenzo; Angelini, Claudia; Carfora, Maria Francesca; Carriero, Maria Vincenza; Natalini, Roberto

    Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences.

  2. Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay.

    Wollin, Klaus-M; Gorlitz, Bernd-D


    The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

  3. A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion

    Neufeld Gera


    Full Text Available Abstract Background The induction of tumor cell invasion is an important step in tumor progression. Due to the cost and slowness of in-vivo invasion assays, there is need for quantitative in-vitro invasion assays that mimic as closely as possible the tumor environment and in which conditions can be rigorously controlled. Methods We have established a novel asymmetric 3D in-vitro invasion assay by embedding a monolayer of tumor cells between two layers of collagen. The cells were then allowed to invade the upper and lower layers of collagen. To visualize invading cells the gels were sectioned perpendicular to the monolayer so that after seeding the monolayer appears as a thin line precisely defining the origin of invasion. The number of invading tumor cells, their proliferation rate, the distance they traverse and the direction of invasion could then be determined quantitatively. Results The assay was used to compare the invasive properties of several tumor cell types and the results compare well with those obtained by previously described assays. Lysyl-oxidase like protein-2 (Loxl2 is a potent inducer of invasiveness. Using our assay we show for the first time that inhibition of endogenous Loxl2 expression in several types of tumor cells strongly inhibits their invasiveness. We also took advantage of the asymmetric nature of the assay in order to show that fibronectin enhances the invasiveness of breast cancer cells more potently than laminin. The asymmetric properties of the assay were also used to demonstrate that soluble factors derived from fibroblasts can preferentially attract invading breast cancer cells. Conclusion Our assay displays several advantages over previous invasion assays as it is allows the quantitative analysis of directional invasive behavior of tumor cells in a 3D environment mimicking the tumor microenvironment. It should be particularly useful for the study of the effects of components of the tumor microenvironment on

  4. Rationale for the real-time and dynamic cell death assays using propidium iodide

    Zhao, Hong; Oczos, Jadwiga; Janowski, Pawel; Trembecka, Dominika; Dobrucki, Jurek; Darzynkiewicz, Zbigniew; Wlodkowic, Donald


    We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. The present study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long term proliferation capacity, DNA damage response and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and las...

  5. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman


    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  6. A high-throughput assay of NK cell activity in whole blood and its clinical application

    Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)


    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  7. Liposomes and MTT cell viability assay: an incompatible affair.

    Angius, Fabrizio; Floris, Alice


    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to evaluate the cytotoxicity potential of drugs vehicled by liposomes. However, liposome delivering drugs could produce inconsistent values of MTT absorbance. On the basis of previous experiments demonstrating the MTT affinity for lipid droplets, this paper aims to show that empty-liposomes interfere, per se, on MTT assay due to its lipidic nature. This brings into question the use of MTT testing cytotoxicity when liposomes are involved in delivering drugs.

  8. Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

    Zhi-hui Huang; Ying Wang; Li Cao; Zhi-da Su; Yan-ling Zhu; Yi-zhang Chen; Xiao-bing Yuan; Cheng He


    Olfactory ensheathing cells (OECs) are a unique type of glial cells that have axonal growth-promoting properties. OEC transplantation has emerged as a promising experimental therapy of axonal injuries and demyelinating diseases. However, some fundamental cellular properties of OECs remain unclear. In this study, we found that the distinct OEC subpopulations exhibited different migratory properties based on time-lapse imaging of single isolated cells, possibly due to their different cytoskeletal organizations. Moreover, OEC subpopulations displayed different attractive migratory responses to a gradient of lysophosphatidic acid (LPA) in single-cell migration assays. Finally, we found that OEC subpopulations transformed into each other spontaneously. Together, these results demonstrate, for the first time to our knowledge, that distinct OEC subpopulations display different migratory properties in vitro and provide new evidence to support the notion of OECs as a single cell type with malleable functional phenotypes.

  9. In vitro assays for cobblestone area-forming cells, LTC-IC, and CFU-C.

    van Os, Ronald P; Dethmers-Ausema, Bertien; de Haan, Gerald


    Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone area-forming cell assay; CAFC). These assays measure the potential of a test cell population retrospectively, i.e., at the time its activity is evident when the stem cell itself is often not detectable anymore. Although the in vitro LTC-IC and CAFC assays have been shown to correlate with in vivo activity, in vivo transplantation assays, where it can be shown that cells possess the ability to indefinitely repopulate all blood lineages, are the ultimate proof for HSC activity. Nevertheless, these in vitro assays provide an excellent method to screen for stem cell activity of a putative stem cell population or for screening the effect of a certain treatment on HSCs.

  10. Inhibition of neuronal cell-cell adhesion measured by the microscopic aggregation assay and impedance sensing

    Wiertz, R. W. F.; Marani, E.; Rutten, W. L. C.


    Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron-neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron-neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control of spatial arrangement of cells in culture. In the literature N-CAM, L1 and N-cadherin proteins are pointed out as main regulators of neuronal adhesion. In this study, these three main cell adhesion molecules were used to inhibit neuron-to-neuron adhesion and aggregation. Both soluble extracellular domains and antigen antibodies were added to these adhesion molecules. They were investigated for their blocking ability in neuronal cultures. First, in a 96 h aggregation assay on a low-adhesive substrate, the effect of inhibition of the three proteins on aggregation of cortical neurons was investigated optically. Both L1 antibody and L1 protein had no effect on the degree of aggregation. An N-cadherin antibody however was shown to be effective in aggregation inhibition at concentrations of 1 and 3 µg ml-1. Up to 96 h no aggregation occurred. A similar effect was achieved by the N-cadherin protein, although less distinct. N-CAM blocking revealed no inhibition of aggregation. Second, results from IS corresponded to those of the aggregation assays. In these experiments neuron-neuron adhesion was also inhibited by blocking N-CAM L1 and N-cadherin. Cortical neurons were cultured in small wells containing circular 100 µm diameter gold electrodes, so small changes in cell-cell interactions in monolayers of neurons could be monitored by IS. Impedances of neuron-covered electrodes were significantly lower in the presence of the N-cadherin antibody and protein at concentrations of 1, 3 and 10 µg ml-1, indicating a less profound binding between adjacent neurons. Results from the aggregation assays and impedance measurements demonstrate the applicability of blocking cell adhesion

  11. Determination of Metabolic Viability and Cell Mass Using a Tandem Resazurin/Sulforhodamine B Assay.

    Silva, Filomena S G; Starostina, Irina G; Ivanova, Vilena V; Rizvanov, Albert A; Oliveira, Paulo J; Pereira, Susana P


    The identification of rapid, reliable, and highly reproducible biological assays that can be standardized and routinely used in preclinical tests constitutes a promising approach to reducing drug discovery costs and time. This unit details a tandem, rapid, and reliable cell viability method for preliminary screening of chemical compounds. This assay measures metabolic activity and cell mass in the same cell sample using a dual resazurin/sulforhodamine B assay, eliminating the variation associated with cell seeding and excessive manipulations in assays that test different cell samples across plates. The procedure also reduces the amount of cells, test compound, and reagents required, as well as the time expended in conventional tests, thus resulting in a more confident prediction of toxic thresholds for the tested compounds. © 2016 by John Wiley & Sons, Inc.

  12. Processing of nanolitre liquid plugs for microfluidic cell-based assays

    Junji Fukuda, Shintaro Takahashi, Tatsuya Osaki, Naoto Mochizuki and Hiroaki Suzuki


    Full Text Available Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

  13. A Cell Lysis and Protein Purification - Single Molecule Assay Devices for Evaluation of Genetically Engineered Proteins

    Nakyama, Tetsuya; Tabata, Kazuhito; Noji, Hiroyuki; Yokokawa, Ryuji

    We have developed two devices applicable to evaluate genetically engineered proteins in single molecule assay: on-chip cell lysis device, and protein purification - assay device. A motor protein, F1-ATPase expressed in E.coli, was focused in this report as a target protein. Cell lysis was simply performed by applying pulse voltage between Au electrodes patterned by photolithography, and its efficiency was determined by absorptiometry. The subsequent processes, purification and assay of extracted proteins, were demonstrated in order to detect F1-ATPase and to evaluate its activity. The specific bonding between his-tag in F1-ATPase and Ni-NTA coated on a glass surface was utilized for the purification process. After immobilization of F1-ATPase, avidin-coated microspheres and adenosine tri-phosphate (ATP) solution were infused sequentially to assay the protein. Microsphere rotation was realized by activity of F1-ATPase corresponding to ATP hydrolysis. Results show that the cell lysis device, at the optimum condition, extracts enough amount of protein for single molecule assay. Once cell lysate was injected to the purification - assay device, proteins were diffused in the lateral direction in a Y-shape microchannel. The gradient of protein concentratioin provides an optimal concentration for the assay i.e. the highest density of rotating beads. Density of rotating beads is also affected by the initial concentration of protein injected to the device. The optimum concentration was achieved by our cell lysis device not by the conventional method by ultrasonic wave. Rotation speed was analyzed for several microspheres assayed in the purification - assay device, and the results were compatible to that of conventional assay in which F1-ATPase was purified in bulk scale. In conclusion, we have demonstrated on-chip cell lysis and assay appropriate for the sequential analysis without any pretreatment. On-chip devices replacing conventional bioanalytical methods will be

  14. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can rec...

  15. Modification of the diphenylamine assay for cell quantification in three-dimensional biodegradable polymeric scaffolds.

    Pham, Edward A; Ho, Won Jin; Kamei, Daniel T; Wu, Benjamin M


    As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects. Thus, additional steps are required to ensure complete isolation of cells from the 3D scaffold. The diphenylamine assay isolates cellular DNA, degrades the polymeric matrix materials, and reacts with the DNA to yield a colorimetric response. Thus, we report here a practical modification of the diphenylamine assay and show that the assay quantifies cells in 3D polyester scaffolds reliably and reproducibly as long as the necessary amount of the acidic working reagent is present. Our study also demonstrates that the sensitivity of the assay can be optimized by controlling the dimensions of the sampling volume. Overall, the DPA assay offers an attractive solution for challenges associated with 3D cell quantification.

  16. Single cell adhesion assay using computer controlled micropipette.

    Rita Salánki

    Full Text Available Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day. Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min. We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a

  17. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening.

    Duellman, Sarah J; Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta


    Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z' = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples.

  18. Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

    Tanneberger, K.; Rico Rico, A.; Kramer, N.I.; Busser, F.J.M.; Hermens, J.L.M.; Schirmer, K.


    Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly underst

  19. Functional screening with a live cell imaging-based random cell migration assay.

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob


    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  20. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G


    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  1. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    Møller-Larsen, A; Brudek, T; Petersen, T


    as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted......Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable...... assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients...

  2. Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion.

    Nakane, Shuhei; Matsuda, Zene


    Fusion between viral and cellular membranes is the essential first step in infection of enveloped viruses. This step is mediated by viral envelope glycoproteins (Env) that recognize cellular receptors. The membrane fusion between the effector cells expressing viral Env and the target cells expressing its receptors can be monitored by several methods. We have recently developed a pair of chimeric reporter protein composed of split Renilla luciferase (RL) and split GFP. We named this reporter dual split protein (DSP), since it recovers both RL and GFP activities upon self reassociation. By using DSP, pore formation and content mixing between the effector and target cells can be monitored upon the recovery of RL and GFP activities after the membrane fusion. This quick assay provides quantitative as well as spatial information about membrane fusion mediated by viral Env.

  3. Polyacrylamide gels for invadopodia and traction force assays on cancer cells.

    Jerrell, Rachel J; Parekh, Aron


    Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user's specific biological and technical needs.

  4. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    Jiang, Shibo; Radigan, Lin; Zhang, Li


    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  5. Adapted cytokinesis-block micronucleus assay (CBMn) for mouse embryonic stem cells



    Authors: Hamid Kalantari, Hamid Gourabi & Hossein Baharvand ### Abstract Our observation showed the addition of cytochalasin-B to mouse embryonic stem cells (mESC) culture for CBMn analysis led to the induction of apoptosis in these cells. On the other hand, addition of cyt-B is the most critical part of the cytokinesis-block micronucleus assay (CBMn) technique that cannot be omitted. Thus, modification of the traditional CBMn assay seems to be necessary. In this paper, we attempt...

  6. Microinjection and Fluorescence In Situ Hybridization Assay for Studying mRNA Export in Mammalian Cells.

    Wang, Ke; Shi, Min; Cheng, Hong


    Microinjection and Fluorescence in situ Hybridization (FISH) assay is a useful method for mRNA export studies, which can overcome the problems of traditional transfection in cells. Here, we describe the method of microinjection and FISH assay applied in investigation of mRNA export. By this method we can estimate the mRNA export kinetics, examining mRNA export in cells with low transfection efficiencies, and observing nuclear export of aberrant RNAs.

  7. Predictive value of cell assays for developmental toxicity and embryotoxicity of conazole fungicides.

    Sørensen, Karin Dreisig; Taxvig, Camilla; Kjærstad, Mia Birkhøj; Nellemann, Christine Lydia; Hass, Ulla; Vinggaard, Anne Marie


    This paper evaluates in vivo predictability of a battery of in vitro tests covering developmental toxicity and embryotoxicity of five widely used conazole fungicides. The conazoles were investigated in the embryonic stem cell test, and data were compared to in vivo embryotoxicity data. The same conazoles were evaluated on the basis of data from a battery of cell assays for endocrine activity, including assays for AR, ER, AhR, and sex hormone synthesis, and data were compared to in vivo develo...

  8. Resazurin Live Cell Assay: Setup and Fine-Tuning for Reliable Cytotoxicity Results.

    Rodríguez-Corrales, José Á; Josan, Jatinder S


    In vitro cytotoxicity tests allow for fast and inexpensive screening of drug efficacy prior to in vivo studies. The resazurin assay (commercialized as Alamar Blue(®)) has been extensively utilized for this purpose in 2D and 3D cell cultures, and high-throughput screening. However, improper or lack of assay validation can generate unreliable results and limit reproducibility. Herein, we report a detailed protocol for the optimization of the resazurin assay to determine relevant analytical (limits of detection, quantification, and linear range) and biological (growth kinetics) parameters, and, thus, provide accurate cytotoxicity results. Fine-tuning of the resazurin assay will allow accurate and fast quantification of cytotoxicity for drug discovery. Unlike more complicated methods (e.g., mass spectrometry), this assay utilizes fluorescence spectroscopy and, thus, provides a less costly alternative to observe changes in the reductase proteome of the cells.

  9. Capacitance-based assay for real-time monitoring of endocytosis and cell viability.

    Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa


    Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.

  10. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay.

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K


    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  11. Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

    Jianwei ZHEN; Qincl LIU; Jianhong GU; Yan YUAN; Xuezhong LIU; Handong WANG; Zongping LIU; Jianchun BIAN


    Abstract [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD~o) of ZEN with tetra- zolium-based colorimetric assay (MTT assay). Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group (P〈0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60_+4.78, 57.75_+6.25, 78.97_+5.83, 100.50~6.94 and 146.83_+12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_+2.25%, 22.24_+2.43%, 31.21_+ 6.27%, 37.45_+4.33% and 60.68_+9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences (P〈0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

  12. Radiotoxicity induced by Auger electron emitters in human osteosarcoma cell line using comet assay

    XU Yu-Jie; LI Qing-Nuan; ZHU Ran; ZHU Ben-Xing; ZHANG Yong-Ping; ZHANG Xiao-Dong; FAN Wo; HONG Cheng-Jiao; LI Wen-Xin


    The comet assay (single cell gel electrophoresis assay) was used to evaluate the radiotoxicity of Augerelectron emitters in the human osteosarcoma cell line (HOS-8603). After internal exposure to 67Ga-EDTMP, the sar-coma cell has been injured severely. The comet length was longer along with the increase of dose, the appearance ofcomet tail was different from that with respect to the 60Co γ-ray irradiation. DNA damage of cell was mainly due tothe radiation effect of Auger electrons. The 67Ga may be a therapeutic radionuclide with good prospect for tumortreatment and palliation of bone pain induced by metastasis.

  13. Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa.

    Sheng, Y; Pero, R W; Amiri, A; Bryngelsson, C


    Growth inhibitory activities of novel water extracts of Uncaria tomentosa (C-Med-100) were examined in vitro using two human leukemic cell lines (K562 and HL60) and one human EBV-transformed B lymphoma cell line (Raji). The proliferative capacities of HL60 and Raji cells were strongly suppressed in the presence of the C-Med-100 while K562 was more resistant to the inhibition. Furthermore, the antiproliferative effect was confirmed using the clonogenic assay, which showed a very close correlation between C-Med-100 concentration and the surviving fraction. The suppressive effect of Uncaria tomentosa extracts on tumor cell growth appears to be mediated through induction of apoptosis which was demonstrated by characteristic morphological changes, internucleosomal DNA fragmentation after agarose gel electrophoresis and DNA fragmentation quantification. C-Med-100 induced a delayed type of apoptosis becoming most dose-dependently prominent after 48 hours of exposure. Both DNA single and double strand breaks were increased 24 hours after C-Med-100 treatment, which suggested a well-established linkage between the DNA damage and apoptosis. The induction of DNA strand breaks coupled to apoptosis may explain the growth inhibition of the tumor cells by Uncaria tomentosa extracts. These results provide the first direct evidence for the antitumor properties of Uncaria tomentosa extracts to be via a mechanism of selective induction of apoptosis.

  14. Novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics but with combinations and probability: bystanders in bulk effector cells influence results of cell-mediated cytotoxicity assays.

    Takayanagi, Toshiaki


    Cell-mediated cytotoxicity assays are widely implemented to evaluate cell-mediated cytotoxic activity, and some assays are analyzed using the analogy of enzyme kinetics. In the analogy, the effector cell is regarded as the enzyme, the target cell as the substrate, the effector cell-target cell conjugate as the enzyme-substrate complex and the dead target cell as the product. However, the assumptions analogous to those of enzyme kinetics are not always true in cell-mediated cytotoxicity assays, and the parameter analogous to the Michaelis-Menten constant is not constant but is dependent on the number of effector cells. Therefore I present novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics. I instead use combinations and probability, because analysis of cell-mediated cytotoxicity assays by applying enzyme kinetics seems controversial. With my original models, I demonstrate simulations of the data in previously published papers. The results are exhibited in the same forms as the corresponding data. Comparing the simulation results with the published data, the results seem to agree well with the data. From simulations of cytotoxic assays with bulk effector cells, it appears that bystanders in bulk effector cells increase both the cytotoxic activity and the motility of effector cells.

  15. Enumeration of Neural Stem Cells Using Clonal Assays.

    Narayanan, Gunaseelan; Yu, Yuan Hong; Tham, Muly; Gan, Hui Theng; Ramasamy, Srinivas; Sankaran, Shvetha; Hariharan, Srivats; Ahmed, Sohail


    Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages - astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

  16. High-throughput fluorescent-based NKCC functional assay in adherent epithelial cells.

    Carmosino, Monica; Rizzo, Federica; Torretta, Silvia; Procino, Giuseppe; Svelto, Maria


    The kidney-specific NKCC cotransporter isoform NKCC2 is involved in the Na(+) reabsorption in the Thich Ascending Limb (TAL) cells and in the regulation of body fluid volume. In contrast, the isoform NKCC1 represents the major pathway for Cl- entry in endothelial cells, playing a crucial role in cell volume regulation and vascular tone. Importantly, both NKCC isoforms are involved in the regulation of blood pressure and represent important potential drug targets for the treatment of hypertension. Taking advantage of an existing Thallium (Tl(+))-based kit, we set up a Tl(+) influx-based fluorescent assay, that can accurately and rapidly measure NKCC transporter activity in adherent epithelial cells using the high-throughput Flex station device. We assessed the feasibility of this assay in the renal epithelial LLC-PK1 cells stably transfected with a previously characterized chimeric NKCC2 construct (c-NKCC2). We demonstrated that the assay is highly reproducible, offers high temporal resolution of NKCC-mediated ion flux profiles and, importantly, being a continuous assay, it offers improved sensitivity over previous endpoint NKCC functional assays. So far the screening of NKCC transporters activity has been done by (86)Rb(+) influx assays. Indeed, a fluorescence-based high-throughput screening method for testing NKCC inhibitors would be extremely useful in the development and characterization of new anti-hypertensive drugs.

  17. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned.

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile


    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  18. Natural killer cell cytotoxicity assay with time-resolved fluorimetry

    李建中; 章竹君; 金伯泉; 田方


    A new time-resolved fluorimetric method for the measurement of natural killer (NK) cell cytotoxicity has been developed by labelling the target cell K562 with a new synthesized fluorescence marker KLUK. The method has advantages of higher sensitivity, time-saving, good reproducibility and has no radioactivity problems. A satisfactory result is obtained by comparing it with 51Cr release method. It demonstrates that the new marker provides an alternative to currently used radioactive markers for the assessment of in vitro cellular cytotoxicity.

  19. Use of a soluble tetrazolium/formazan assay for chicken cells.

    Tajima, T; Hironao, T; Kajikawa, T; Kawamura, H


    We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.

  20. Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

    Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine


    Selective serotonin reuptake inhibitors are known to have a range of disorders that are often linked to the endocrine system e.g. hormonal imbalances, breast enlargement, sexual dysfunction, and menstrual cycle disorders. The mechanisms behind most of these disorders are not known in details....... In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were......M for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine...

  1. Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.

    Silvia Blacher

    Full Text Available The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph angiogenesis and test pro- and anti-(lymph angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

  2. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung


    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

  3. Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

    Phairote Teeranaipong


    Full Text Available Introduction: A dual split reporter protein system (DSP, recombining Renilla luciferase (RL and green fluorescent protein (GFP split into two different constructs (DSP1–7 and DSP8–11, was adapted to create a novel rapid phenotypic tropism assay (PTA for HIV-1 infection (DSP-Pheno. Methods: DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4 or CD4/CCR5 (N4R5, respectively. An expression vector with DSP8–11 (pRE11 was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results: The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP. The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions: A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and

  4. Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

    Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel


    The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

  5. Comparison between fibroblast wound healing and cell random migration assays in vitro.

    Ascione, Flora; Vasaturo, Angela; Caserta, Sergio; D'Esposito, Vittoria; Formisano, Pietro; Guido, Stefano


    Cell migration plays a key role in many biological processes, including cancer growth and invasion, embryogenesis, angiogenesis, inflammatory response, and tissue repair. In this work, we compare two well-established experimental approaches for the investigation of cell motility in vitro: the cell random migration (CRM) and the wound healing (WH) assay. In the former, extensive tracking of individual live cells trajectories by time-lapse microscopy and elaborate data processing are used to calculate two intrinsic motility parameters of the cell population under investigation, i.e. the diffusion coefficient and the persistence time. In the WH assay, a scratch is made in a confluent cell monolayer and the closure time of the exposed area is taken as an easy-to-measure, empirical estimate of cell migration. To compare WH and CRM we applied the two assays to investigate the motility of skin fibroblasts isolated from wild type and transgenic mice (TgPED) overexpressing the protein PED/PEA-15, which is highly expressed in patients with type 2 diabetes. Our main result is that the cell motility parameters derived from CRM can be also estimated from a time-resolved analysis of the WH assay, thus showing that the latter is also amenable to a quantitative analysis for the characterization of cell migration. To our knowledge this is the first quantitative comparison of these two widely used techniques. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Patterning cell using Si-stencil for high-throughput assay

    Wu, Jinbo


    In this communication, we report a newly developed cell pattering methodology by a silicon-based stencil, which exhibited advantages such as easy handling, reusability, hydrophilic surface and mature fabrication technologies. Cell arrays obtained by this method were used to investigate cell growth under a temperature gradient, which demonstrated the possibility of studying cell behavior in a high-throughput assay. This journal is © The Royal Society of Chemistry 2011.

  7. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Straub, Tim M.; Honer Zu Bentrup, Kerstin A.; Orosz Coghlan, Patricia A.; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza; Nickerson, Cheryl A.


    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  8. Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

    Daniel C Farley


    Full Text Available It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02. VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

  9. Ovine carotid artery-derived cells as an optimized supportive cell layer in 2-D capillary network assays.

    Stefan Weinandy

    Full Text Available BACKGROUND: Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. METHODS AND RESULTS: Human umbilical artery SMCs (HUASMCs and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs. Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab, had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. CONCLUSIONS: Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast

  10. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Yomo Tetsuya


    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  11. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  12. Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease

    Li, Quan; Henry, Eric R.; Hofrichter, James; Smith, Jeffrey F.; Cellmer, Troy; Dunkelberger, Emily B.; Metaferia, Belhu B.; Jones-Straehle, Stacy; Boutom, Sarah; Christoph, Garrott W.; Wakefield, Terri H.; Link, Mary E.; Staton, Dwayne; Vass, Erica R.; Miller, Jeffery L.; Hsieh, Matthew M.; Tisdale, John F.; Eaton, William A.


    Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort (“sickle”) the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms. PMID:28096387

  13. Multiple MTS Assay as the Alternative Method to Determine Survival Fraction of the Irradiated HT-29 Colon Cancer Cells.

    Arab-Bafrani, Zahra; Shahbazi-Gahrouei, Daryoush; Abbasian, Mahdi; Fesharaki, Mehrafarin


    A multiple colorimetric assay has been introduced to evaluate the proliferation and determination of survival fraction (SF) of irradiated cells. The estimation of SF based on the cell-growth curve information is the major advantage of this assay. In this study, the utility of multiple-MTS assay for the SF estimation of irradiated HT-29 colon cancer cells, which were plated before irradiation, was evaluated. The SF of HT-29 colon cancer cells under irradiation with 9 MV photon was estimated using multiple-MTS assay and colony assay. Finally, the correlation between two assays was evaluated. Results showed that there are no significant differences between the SF obtained by two assays at different radiation doses (P > 0.05), and the survival curves have quite similar trends. In conclusion, multiple MTS-assay can be a reliable method to determine the SF of irradiated colon cancer cells that plated before irradiation.

  14. Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

    Sandra A. Calarota


    Full Text Available The enzyme-linked immunospot (ELISPOT assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT or after their expansion during a 10-day culture (cultured ELISPOT. Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field.

  15. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane


    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  16. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  17. Identifying intracellular sites of eicosanoid lipid mediator synthesis with EicosaCell assays.

    Bandeira-Melo, Christianne; Weller, Peter F; Bozza, Patricia T


    Eicosanoids, arachidonic acid-derived signaling lipid mediators, are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. EicosaCell is a microscopic assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell is a versatile assay which allows analyses of different types of cell preparations, such as cells isolated from humans or harvested cells from in vivo models of inflammation and adherent or suspension cells stimulated in vitro. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This is of particular interest given that over the past decade intracellular compartmentalization of eicosanoid-synthetic machinery has emerged both as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. This review covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.

  18. Inhibition of neuronal cell–cell adhesion measured by the microscopic aggregation assay and impedance sensing

    Wiertz, Remy; Marani, Enrico; Rutten, Wim


    Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron–neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron–neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control

  19. In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

    Speksnijder, JE; Hage, WJ; Lanser, PH; Collodi, P; Zivkovic, D


    We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning microscopy

  20. Large-scale prospective T cell function assays in shipped, unfrozen blood samples

    Hadley, David; Cheung, Roy K; Becker, Dorothy J


    around the timing of infant vaccinations. This assay platform and shipping protocol satisfy the criteria for robust and reproducible long-term measurements of human T cell function, comparable to those of established blood biochemical tests. We present a stable technology for prospective disease......-relevant T cell analysis in immunological diseases, vaccination medicine, and measurement of herd immunity....

  1. Neutral red uptake assay for the estimation of cell viability/cytotoxicity.

    Repetto, Guillermo; del Peso, Ana; Zurita, Jorge L


    The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

  2. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  3. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I


    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  4. A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins in human peripheral blood.

    Sun, Peifang; Beckett, Charmagne; Danko, Janine; Burgess, Timothy; Liang, Zhaodong; Kochel, Tadeusz; Porter, Kevin


    Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53-513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0-45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4(+) and CD8(+) T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

  5. Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.

    Li, Jie; Zhang, Donglan; Ward, Kathleen M; Prendergast, George C; Ayene, Iraimoudi S


    Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.

  6. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Gilbert, Daniel F; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E; Boutros, Michael


    Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  7. A novel multiplex cell viability assay for high-throughput RNAi screening.

    Daniel F Gilbert

    Full Text Available Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

  8. A molecular assay for sensitive detection of pathogen-specific T-cells.

    Victoria O Kasprowicz

    Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

  9. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok


    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  10. Application of the comet assay in studies of programmed cell death (PCD in plants

    Maria Charzyńska


    Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

  11. Tumor stem cell assay for detecting metastases of human lung cancer.



    Full Text Available We applied a tumor stem cell assay using an enriched double-layered soft agar system for the detection of metastatic sites of lung cancer. Lung cancer colonies grew from 7 of 10 effusions cytologically positive for tumor cells and 7 of 10 bone marrow aspirates cytologically and histologically positive for tumor cells. Twenty-six of 29 bone marrow aspirates cytologically and histologically negative for tumor cells showed no colony growth. However, the remaining three bone marrow aspirates, which were obtained from patients with small cell lung cancer, formed colonies in soft agar. These results indicate that the tumor stem cell assay is useful for detecting metastatic sites of lung cancer.

  12. Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay

    Li-Mei Qiu; Wen-Jian Li; Xin-Yue Pang; Qing-Xiang Gao; Yan Feng; Li-Bin Zhou; Gao-Hua Zhang


    AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft fur Schwerionenforschung mbH, Darmstadt, Germany),remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, γ-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions.METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80Mev/u 20Ne10+ on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy,and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis.RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5Gy to 8Gy (t-test: P<0.001, vs control).The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2Gy, nearly 100 % cells were damaged. Furthermore, both tail length and tail moment, showed linear equation.CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to 20Ne10+.Different reactions

  13. Predictive value of cell assays for developmental toxicity and embryotoxicity of conazole fungicides.

    Dreisig, Karin; Taxvig, Camilla; Birkhøj Kjærstad, Mia; Nellemann, Christine; Hass, Ulla; Vinggaard, Anne Marie


    This paper evaluates in vivo predictability of a battery of in vitro tests covering developmental toxicity and embryotoxicity of five widely used conazole fungicides. The conazoles were investigated in the embryonic stem cell test, and data were compared to in vivo embryotoxicity data. The same conazoles were evaluated on the basis of data from a battery of cell assays for endocrine activity, including assays for AR, ER, AhR, and sex hormone synthesis, and data were compared to in vivo developmental toxicity data. Overall, the ranking of the five conazole fungicides based on in vitro data were in reasonably good agreement with available in vivo effects. Ketoconazole and epoxiconazole are the most potent embryotoxic compounds, whereas prochloraz belongs to the most potent developmental toxicants. In conclusion, a rough prediction of the ranking of these conazole fungicides for in vivo toxicity data was possible by a holistic evaluation of data from a panel of cell-based assays.

  14. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R


    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  15. Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

    Speit, Günter; Schütz, Petra; Bausinger, Julia


    The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.

  16. Genotoxicity testing of PLGA-PEO nanoparticles in TK6 cells by the comet assay and the cytokinesis-block micronucleus assay.

    Kazimirova, Alena; Magdolenova, Zuzana; Barancokova, Magdalena; Staruchova, Marta; Volkovova, Katarina; Dusinska, Maria


    The in vitro genotoxicity of PLGA-PEO (poly-lactic-co-glycolic acid-polyethylene oxide copolymer) nanoparticles was assessed in TK6 cells using the comet assay as well as cytokinesis-block micronucleus (CBMN) assay. The cells were exposed to 0.12-75μg/cm² of PLGA-PEO nanoparticles during 2 and 24h for analysis in the comet assay, and to 3-75μg/cm² of these nanoparticles during 4, 24, 48 and 72h, respectively, for analysis in the CBMN assay. Two different protocols for treatment with cytochalasin B were used. We found that PLGA-PEO was neither cytotoxic (measured by relative cell growth activity and cytokinesis-block proliferation index (CBPI)), nor did it induce DNA strand-breaks (detected by the comet assay) or oxidative DNA lesions (measured by the comet assay modified with lesion-specific enzyme formamidopyrimidine-DNA-glycosylase). There were no statistically significant differences in the frequencies of micronucleated binucleated cells (MNBNCs) between untreated and treated cells in either of the conditions used. This suggests that PLGA-PEO did not have potential genotoxicity. However, using two experimental protocols of the micronucleus assay, PLGA-PEO nanoparticles showed a weak but significant increase in the level of MN in mononucleated cells, in cells treated for 48h with PLGA-PEO nanoparticles when cytochalasin B was added for the last 24h (1st protocol), and in cells treated for 24h with PLGA-PEO nanoparticles followed by washing of NPs and addition of cytochalasin B for another 24h (2nd protocol). It remains unclear whether the increase of MNMNC after treatment with PLGA-PEO nanoparticles is the effect of a possible, weak aneugenic potential or early effect of these particles, or due to another reason. These results suggest that aneugenicity in addition to clastogenicity may be considered as an important biomarker when assessing the genotoxic potential of polymeric nanoparticles.

  17. Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

    McGowan, Eileen M; Alling, Nikki; Jackson, Elise A; Yagoub, Daniel; Haass, Nikolas K; Allen, John D; Martinello-Wilks, Rosetta


    Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the

  18. Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

    Eileen M McGowan

    Full Text Available Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTS assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex, FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle

  19. Application of the ADVIA cerebrospinal fluid assay to count residual red blood cells in blood components.

    Culibrk, B; Stone, E; Levin, E; Weiss, S; Serrano, K; Devine, D V


    There is no automated, accurate assay for the enumeration of residual red blood cells (rRBCs) in non-RBC components for transfusion, despite the potential risk of allo-immunization when mismatched components are transfused. The automated ADVIA 120 cerebrospinal fluid (CSF) assay, which is approved to count RBCs and WBCs in CSF samples, was optimized and tested to measure rRBC in platelet concentrate (PC) and plasma components. Sample dilution, incubation time and reagent volume were optimized for use with non-RBC blood products. The assay was linear (R(2) = 0·99), even at low rRBCs counts. Intra- and inter-assay variation gave coefficients of variance (CV) between 2·2 and 9·4% and 2·6 and 14·9%, respectively, depending on rRBC levels. Good correlation (r = 0·995) was found between the automated assay and manual counting, which is considered the gold standard. Using the automated assay, the range of rRBCs (count/unit) in buffy-coat platelet concentrate (PCs) was 27-5505 × 10(6) and in apheresis PCs was 1-361 × 10(6). The ADVIA CSF assay is a sensitive, precise and accurate means to assess rRBC counts in non-RBC components. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

  20. Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

    de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A


    Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

  1. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    Kwan Yuet Ping; Ibrahim Darah; Yeng Chen; Sreenivasan Sasidharan


    Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

  2. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel


    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  3. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S


    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  4. The loss-of-allele assay for ES cell screening and mouse genotyping.

    Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M


    Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

  5. A simple, versatile and sensitive cell-based assay for prions from various species.

    Zaira E Arellano-Anaya

    Full Text Available Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

  6. Microfluidics meet cell biology: bridging the gap by validation and application of microscale techniques for cell biological assays.

    Paguirigan, Amy L; Beebe, David J


    Microscale techniques have been applied to biological assays for nearly two decades, but haven't been widely integrated as common tools in biological laboratories. The significant differences between several physical phenomena at the microscale versus the macroscale have been exploited to provide a variety of new types of assays (such as gradient production or spatial cell patterning). However, the use of these devices by biologists seems to be limited by issues regarding biological validation, ease of use, and the limited available readouts for assays done using microtechnology. Critical validation work has been done recently that highlights the current challenges for microfluidic methods and suggest ways in which future devices might be improved to better integrate with biological assays. With more validation and improved designs, microscale techniques hold immense promise as a platform to study aspects of cell biology that are not possible using current macroscale techniques.

  7. A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

    Lai, Yiling; Xiang, Meichun; Liu, Shuchun; Li, Erwei; Che, Yongsheng; Liu, Xingzhong


    Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products.

  8. Plaque assay for human coronavirus NL63 using human colon carcinoma cells

    Drosten Christian


    Full Text Available Abstract Background Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. Results 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2. CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. Conclusion CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

  9. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan


    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  10. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan


    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  11. In vitro measurement of cell death with the annexin A5 affinity assay.

    van Genderen, Hugo; Kenis, Heidi; Lux, Petra; Ungeth, Lisette; Maassen, Cecile; Deckers, Niko; Narula, Jagat; Hofstra, Leo; Reutelingsperger, Chris


    One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.

  12. In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

    Pratt, R M; Willis, W D


    We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo. Images PMID:3862095

  13. In vitro screening assay for teratogens using growth inhibition of human embryonic cells

    Pratt, R.M.; Willis, W.D.


    The authors have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. The authors suggest that this assay, along with the complementary tumor cell-attachment assay of Braun may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

  14. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne


    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  15. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Langer, Gernot


    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  16. A simple technique for reducing edge effect in cell-based assays.

    Lundholt, Betina Kerstin; Scudder, Kurt M; Pagliaro, Len


    Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.

  17. Conference report: the 5th cell-based assay and bioanalytical method development conference.

    Ma, Mark


    Approximately 80 participants met at the Marriot Hotel, San Francisco, CA, USA, between the 4th and 6th October 2010 to share novel techniques and discuss the emerging approaches in the evolving field of cell-based assay and bioanalytical method development. This report highlights the discussion and summary of the meeting.

  18. An exo-cell assay for examining real-time γ-secretase activity and inhibition

    Tian Yuan


    Full Text Available Abstract γ-Secretase is an aspartyl protease that cleaves multiple substrates that are involved in broad biological processes ranging from stem cell development to neurodegeneration. The investigation of γ-secretase has been limited by currently available assays that require genetic or biochemical manipulation in the form of substrate transfection or membrane preparation. Here we report an exo-cell assay that is capable of characterizing γ-secretase activity in any cellular system without limitation. Using a highly active, recombinant substrate this assay can quickly and easily ascertain the status of γ-secretase activity in cell systems and patient samples. We have applied this method to determine the activity of γ-secretase in primary cell samples where transfection and/or membrane isolation are not viable options. Importantly, it allows for the detection of real time γ-secretase activity after inhibitor or drug treatment. The application of this assay to determine the role of γ-secretase in physiological and pathological conditions will greatly facilitate our characterization of this complex protease and help in the development and evaluation of γ-secretase-targeted therapies in Alzheimer's disease or a variety of neoplasms.

  19. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors.

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B


    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  20. A microfluidic wound-healing assay for quantifying endothelial cell migration

    van der Meer, Andries Dirk; Vermeul, Kim; Poot, Andreas A.; Feijen, Jan; Vermes, I.

    A microfluidic wound-healing assay for quantifying endothelial cell migration. Am J Physiol Heart Circ Physiol 298: H719–H725, 2010. First published November 20, 2009; doi:10.1152/ajpheart.00933.2009.—Endothelial migration is an important process in the formation of blood vessels and the repair of

  1. Evaporative edge lithography of a liposomal drug microarray for cell migration assays

    Vafai, Nicholas; Lowry, Troy W.; Wilson, Korey A.; Davidson, Michael W.; Lenhert, Steven


    Lipid multilayer microarrays are a promising approach to miniaturize laboratory procedures by taking advantage of the microscopic compartmentalization capabilities of lipids. Here, we demonstrate a new method to pattern lipid multilayers on surfaces based on solvent evaporation along the edge where a stencil contacts a surface called evaporative edge lithography (EEL). As an example of an application of this process, we use EEL to make microarrays suitable for a cell-based migration assay. Currently existing cell migration assays require a separate compartment for each drug which is dissolved at a single concentration in solution. An advantage of the lipid multilayer microarray assay is that multiple compounds can be tested on the same surface. We demonstrate this by testing the effect of two different lipophilic drugs, Taxol and Brefeldin A, on collective cell migration into an unpopulated area. This particular assay should be scalable to test of 2000 different lipophilic compounds or dosages on a standard microtiter plate area, or if adapted for individual cell migration, it would allow for high-throughput screening of more than 50,000 compounds per plate. PMID:27617264

  2. An in vitro clonogenic assay to assess radiation damage in rat CNS glial progenitor cells

    Maazen, R.W.M. van der; Verhagen, I.; Kogel, A.J. van der (Katholieke Univ., Nijmegen (Netherlands). Inst. of Radiotherapy)


    Normal glial progenitor cells can be isolated from the rat central nervous system (CNS) and cultured in vitro on a monolayer of type-1 astrocytes. These monolayers are able to support and stimulate explanted glial progenitor cells to proliferate. Employing these in vitro interactions of specific glial cell types, an in vivo-in vitro clonogenic assay has been developed. This method offers the possibility to study the intrinsic radiosensitivity, repair and regeneration of glial progenitor cells after in vitro or in vivo irradiation. (author).

  3. [An adenosine triphosphate bioluminescence assay for detecting the number of living cells].

    Liu, S; Peng, Z; Wang, H; Lou, J; He, B; Tang, Q; Qiu, D


    The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.

  4. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S.; Yamamoto, Kyoko; Zervantonakis, Ioannis K.; Sudo, Ryo; Kamm, Roger D.; Chung, Seok


    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel incorporating chambers between surface-accessible microchannels. Using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of experiments can run for up to several weeks. PMID:22678430

  5. Application of the comet assay in studies of programmed cell death (PCD) in plants


    Programmed cell death (PCD) in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis) for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anthe...

  6. Adult neural stem cells from the subventricular zone: a review of the neurosphere assay.

    Gil-Perotín, Sara; Duran-Moreno, María; Cebrián-Silla, Arantxa; Ramírez, Mónica; García-Belda, Paula; García-Verdugo, José Manuel


    The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self-renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC-derived neurospheres (including new ultrastructural data). The controversy regarding the identity and "stemness" of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere-derived cells to the clinical setting.

  7. Acute Shear Stress Direction Dictates Adherent Cell Remodeling and Verifies Shear Profile of Spinning Disc Assays

    Fuhrmann, Alexander; Engler, Adam J.


    Several methods have been developed to quantify population level changes in cell attachment strength given its large heterogeneity. One such method is the rotating disc chamber or “spinning disc” in which a range of shear forces are applied to attached cells to quantify detachment force, i.e. attachment strength, which can be heterogeneous within cell populations. However, computing the exact force vectors that act upon cells is complicated by complex flow fields and variable cell morphologies. Recent observations suggest that cells may remodel their morphology and align during acute shear exposure, but contrary to intuition, shear is not orthogonal to the radial direction. Here we theoretically derive the magnitude and direction of applied shear and demonstrate that cells, under certain physiological conditions, align in this direction within minutes. Shear force magnitude is also experimentally verified which validates that for spread cells shear forces and not torque or drag dominate in this assay, and demonstrates that the applied force per cell area is largely independent of initial morphology. These findings suggest that direct quantified comparison of the effects of shear on a wide array of cell types and conditions can be made with confidence using this assay without the need for computational or numerical modeling. PMID:25619322

  8. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Kim, Jin Kyu


    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  9. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger


    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  10. Promoting Activity of Microcystins Extracted From Waterblooms in SHE Cell Transformation Assay



    Microcystis aeruginosa is the dominant algae in most of the eutrophicated lakes in China.It can produce cyclic heptapeptides,Known as microcystins,which can cause liver damage in wild and comestic animals.In this paper,a two-stage transformation assay for demonstrating the carcinogenic effects of the algan toxins is reported.The cell strain used in this assay was derived from embryos of Syrian golden hamter and the algal toxins were extracted from Microcystis aeruginosa,termed microcystis raw toxic(MRT).To elucidate is prooting activity,the target cells were first exposed to a low dosage of 3-methylcholanthrene(MCA)and then to MRT.The results showed that MRT significantly enhanced the MCA-initiated cell transformation,and a dose-response reltionship was observed,but it failed to induce transformation of SHE cells not pretreated by MCA.These results suggest that the MRT play an important role in the malignant transformation of SHE cells.MRT may thus be a tumor promoter,and this transformation assay with SHE cells may be used to predict tumor prompting activity of environmental chemicals before long-term in vivo two-stage carcinogenesis experiments are carried out.

  11. Cell-patterned glass spray for direct drug assay using mass spectrometry

    Wu, Jing [School of Science, China University of Geosciences (Beijing), Beijing 100083 (China); Wang, Shiqi; Chen, Qiushui [Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 (China); Jiang, Hao; Liang, Shuping [School of Science, China University of Geosciences (Beijing), Beijing 100083 (China); Lin, Jin-Ming, E-mail: [Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084 (China)


    In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R{sup 2} = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

  12. Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots.

    Williams, Y; Byrne, S; Bashir, M; Davies, A; Whelan, A; Gun'ko, Y; Kelleher, D; Volkov, Y


    Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots. Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence. This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.

  13. A highly sensitive cell assay for validation of purification regimes of alginates.

    Leinfelder, U; Brunnenmeier, F; Cramer, H; Schiller, J; Arnold, K; Vásquez, J A; Zimmermann, U


    Among the hydrogels used for microencapsulation of cells and tissues, alginate has been and will continue to be one of the most important biomaterials. A mandatory requirement for clinical immunoisolated transplantations is the reproducible production of biocompatible alginate. As shown here for alginates extracted from freshly collected algal stipes, the current assays used for validation of the quality of the alginate are not sufficient to screen for impurities arising from spores of gram-positive bacteria (and related contaminants). To assess the quality of alginate, we have developed a cell assay based on the induction of apoptosis in Jurkat cells. This assay allows in combination with the "modified mixed lymphocyte" assay a rapid and sensitive screening for any fibrosis-inducing impurities in alginate samples (even during the purification regime) as demonstrated by transplantation experiments performed in parallel with BB rats (exhibiting an elevated macrophage activity). The results clearly demonstrate that the quality of the input algal material is of key relevance for the production of transplantation-grade alginate.

  14. GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

    Beatriz A Santillan

    Full Text Available Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

  15. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik


    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  16. In vitro assay of nuclear uptake of doxorubicin hydrochloride in osteosarcoma cells of dogs.

    Weinstein, M J; Berg, J; Kusuzaki, K; Springfield, D S; Gebhardt, M C; Mankin, H J


    A rapid, simple chemosensitivity assay, assessing tumor cell nuclear uptake of doxorubicin hydrochloride, was evaluated in 16 dogs with appendicular osteosarcoma. Doxorubicin was administered to dogs in 5 biweekly treatments, and surgical resection was performed after the second or third treatment. The chemosensitivity assay was performed on biopsy specimens from all dogs before chemotherapy. It was repeated on tissue from resected tumors, and tumors were evaluated histologically to determine the degree of necrosis resulting from chemotherapy. Disease-free and total survival time correlated significantly (P less than 0.05 in both cases) with the degree of postchemotherapy necrosis of the primary tumors. Significant correlation was not apparent between the percentage of tumor cells with nuclear uptake of doxorubicin (in either biopsy or resection samples) and disease-free or total survival time. The percentage of cells with nuclear uptake of doxorubicin in surgically resected tumors correlated significantly (P less than 0.05) with percentage of necrosis.

  17. Action of tumor initiators and promoters in the Syrian hamster embryo cell transformation assay

    Jones, C.A.; Huberman, E.


    The Syrian hamster embryo (SHE) cell transformation assay is unique among the rodent fibroblast transformation systems in that it uses normal, diploid cells. Alteration in the control of growth in carcinogen-treated cultures is used to indicate the onset of neoplastic development. An evaluation of the SHE assay for screening carcinogens is reported. Using coded chemicals, the degree of intra- and interlaboratory reproducibility with the system was evaluated. Overall, there was a good qualitative correlation between the carcinogenicity of the chemicals and their ability to induce morphological cell transformation. Unfortunately, the low level of response and lack of good dose-response relationships with certain chemical are still major constraints to the use of this system in routine testing. Further consideration needs to be given to developing procedures that select for, or amplify, expression of the transformed phenotype. 9 refs., 2 figs., 1 tab.

  18. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail:; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)


    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  19. Lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay.

    Ribeiro, Daniel Araki; Marques, Mariângela Esther Alencar; Salvadori, Daisy Maria Fávero


    Formocresol, paramonochlorophenol, and calcium hydroxide are widely used in dentistry because of their antibacterial activities in root canal disinfection. However, the results of genotoxicity studies using these materials are inconsistent in literature. The goal of this study was to examine the genotoxic potential of formocresol, paramonochlorophenol, and calcium hydroxide using mouse lymphoma cells and human fibroblasts cells in vitro by the comet assay. Data were assessed by Kruskal-Wallis nonparametric test. The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

  20. Viral glycoprotein-mediated cell fusion assays using vaccinia virus vectors.

    Bossart, Katharine N; Broder, Christopher C


    The vaccinia virus-based expression of viral envelope glycoprotein genes-derived from enveloped viruses that infect their respective host cells through a pH-independent mechanism of membrane fusion-has been a powerful tool in helping to characterize these important attachment and fusion proteins. The cellular expression of these viral envelope glycoproteins has allowed for the measurement of membrane fusion events using cell-cell fusion or syncytia formation. This method has been enhanced by the addition of a reporter-gene system to the vaccinia virus-based cell-cell fusion assay. This improvement has provided a high-throughput and quantitative aspect to this assay, which can serve as a surrogate for virus entry and is therefore ideally suited in the characterization of numerous enveloped viruses, including biological safety level-4 (BSL-4) agents. This chapter will detail the methods of the vaccinia virus-based reporter-gene fusion assay and how it may be used to characterize the fusion mediated by the BSL-4-classified Hendra and Nipah viruses.

  1. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan


    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. One-step seeding of neural stem cells with vitronectin-supplemented medium for high throughput screening assays

    Dai, Sheng; Li, Rong; Long, Yan; Titus, Steve; Zhao, Jinghua; Huang, Ruili; Xia, Menghang; Zheng, Wei


    Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research. PMID:27647668

  3. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

    Kimura, Aoi; Miyata, Atsuro; Honma, Masamitsu


    The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

  4. Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

    Berckmans, P; Leppens, H; Vangenechten, C; Witters, H


    There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

  5. Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

    Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P


    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.

  6. First application of comet assay in blood cells of Mediterranean loggerhead sea turtle (Caretta caretta).

    Caliani, Ilaria; Campani, Tommaso; Giannetti, Matteo; Marsili, Letizia; Casini, Silvia; Fossi, Maria Cristina


    The aim of this study was to validate the comet assay in erythrocytes of Caretta caretta, a species never investigated for genotoxicity. We studied 31 loggerhead sea turtles from three Italian marine rescue centres. Peripheral blood samples were collected from all the animals and the comet assay applied. All comet cells were analysed using two methods: visual scoring and computer image analysis. The % DNA in tail mean value ± SD and Damage Index were 21.56 ± 15.41 and 134.83 ± 94.12, respectively. A strong and statistically significant statistically correlation between the two analytical methods was observed (r = 0.95; p comet assay is a useful method to detect the possible effects of genotoxic agents in loggerhead sea turtle and to increase the knowledge about the ecotoxicological health status of this threatened species.

  7. An introduction to the wound healing assay using live-cell microscopy.

    Jonkman, James E N; Cathcart, Judith A; Xu, Feng; Bartolini, Miria E; Amon, Jennifer E; Stevens, Katarzyna M; Colarusso, Pina


    The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.

  8. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    Mahajan Supriya D.


    Full Text Available Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

  9. Measurement of single-cell adhesion strength using a microfluidic assay.

    Christ, Kevin V; Williamson, Kyle B; Masters, Kristyn S; Turner, Kevin T


    Despite the importance of cell adhesion in numerous physiological, pathological, and biomaterial-related responses, our understanding of adhesion strength at the cell-substrate interface and its relationship to cell function remains incomplete. One reason for this deficit is a lack of accessible experimental approaches that quantify adhesion strength at the single-cell level and facilitate large numbers of tests. The current work describes the design, fabrication, and use of a microfluidic-based method for single-cell adhesion strength measurements. By applying a monotonically increasing flow rate in a microfluidic channel in combination with video microscopy, the adhesion strength of individual NIH3T3 fibroblasts cultured for 24 h on various surfaces was measured. The small height of the channel allows high shear stresses to be generated under laminar conditions, allowing strength measurements on well-spread, strongly adhered cells that cannot be characterized in most conventional assays. This assay was used to quantify the relationship between morphological characteristics and adhesion strength for individual well-spread cells. Cell adhesion strength was found to be positively correlated with both cell area and circularity. Computational fluid dynamics (CFD) analysis was performed to examine the role of cell geometry in determining the actual stress applied to the cell. Use of this method to examine adhesion at the single-cell level allows the detachment of strongly-adhered cells under a highly-controllable, uniform loading to be directly observed and will enable the characterization of biological events and relationships that cannot currently be achieved using existing methods.

  10. Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis.

    Yen, Ching-Yu; Wu, Yu-Wei; Hsiung, Chao-Nan; Yeh, Min-I; Lin, Yi-Ming; Lee, Sheng-Yang


    Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

  11. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

    Kaoru Miyazaki

    Full Text Available BACKGROUND: Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. METHODOLOGY/PRINCIPAL FINDINGS: ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. CONCLUSIONS/SIGNIFICANCE: We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo

  12. Intralaboratory validation of cell-free translation assay for detecting ricin toxin biological activity.

    Lindsey, Changhong Y; Richardson, Jenny D; Brown, J Edward; Hale, Martha L


    A cell-free translation (CFT) assay for determining ricin biological activity was validated. The statistical data from the validation study showed a high level of precision within and between runs of the assay. The assay was specific for determining ricin biological activity in food-based matrixes and discriminated ricin from other ribosome-inactivating proteins. The mean bias (relative error) between measured ricin concentrations of 3 validation samples and their nominal concentrations was 1.1, 6.6, and 20.3%, while the coefficient of variation (CV) was 14.1, 7.7, and 13.5%, respectively, demonstrating good precision, accuracy, and linearity. The CVs of ricin concentrations in 2 ricin-containing samples calculated from a dilution series were <5 and <12%, respectively, demonstrating very good parallelism. The analyte stability of ricin-containing samples stored for 1 month either at 4 or -20 degrees C, the stability of ricin stock solutions, and the results of assays executed by different analysts and using different luminometers were evaluated. The statistical validation data confirmed that the 4-parameter logistic equation, y = (a - d)/[1 + (x/c)b] + d, provided an accurate representation of a sigmoidal relationship between the measured response and the observed ricin concentration for the CFT assay.

  13. In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

    Darne, Christian; Coulais, Catherine; Terzetti, Francine; Fontana, Caroline; Binet, Stéphane; Gaté, Laurent; Guichard, Yves


    Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard

  14. In vitro assay for HCV serine proteinase expressed in insect cells

    Li-Hua Hou; Gui-Xin Du; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang


    AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metalchelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5×107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients′ sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

  15. Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

    Wakuri, S; Yamakage, K; Kazuki, Y; Kazuki, K; Oshimura, M; Aburatani, S; Yasunaga, M; Nakajima, Y


    The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency.

    Polanco, Juan Carlos; Ho, Mirabelle S H; Wang, Bei; Zhou, Qi; Wolvetang, Ernst; Mason, Elizabeth; Wells, Christine A; Kolle, Gabriel; Grimmond, Sean M; Bertoncello, Ivan; O'Brien, Carmel; Laslett, Andrew L


    Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.

  17. Crucial ignored parameters on nanotoxicology: the importance of toxicity assay modifications and "cell vision".

    Sophie Laurent

    Full Text Available Until now, the results of nanotoxicology research have shown that the interactions between nanoparticles (NPs and cells are remarkably complex. In order to get a deep understanding of the NP-cell interactions, scientists have focused on the physicochemical effects. However, there are still considerable debates about the regulation of nanomaterials and the reported results are usually in contradictions. Here, we are going to introduce the potential key reasons for these conflicts. In this case, modification of conventional in vitro toxicity assays, is one of the crucial ignored matter in nanotoxicological sciences. More specifically, the conventional methods neglect important factors such as the sedimentation of NPs and absorption of proteins and other essential biomolecules onto the surface of NPs. Another ignored matter in nanotoxicological sciences is the effect of cell "vision" (i.e., cell type. In order to show the effects of these ignored subjects, we probed the effect of superparamagnetic iron oxide NPs (SPIONs, with various surface chemistries, on various cell lines. We found thatthe modification of conventional toxicity assays and the consideration of the "cell vision" concept are crucial matters to obtain reliable, and reproducible nanotoxicology data. These new concepts offer a suitable way to obtain a deep understanding on the cell-NP interactions. In addition, by consideration of these ignored factors, the conflict of future toxicological reports would be significantly decreased.

  18. A parallel and quantitative cell migration assay using a novel multi-well-based device.

    Quan, Qianghua; Zhang, Shuwen; Wang, Xudong; Ouyang, Qi; Wang, Yugang; Yang, Gen; Luo, Chunxiong


    Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-β(TGF-β) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-β + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

  19. A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells.

    Noiman, Tsahi; Buzhor, Ella; Metsuyanim, Sally; Harari-Steinberg, Orit; Morgenshtern, Chaya; Dekel, Benjamin; Goldstein, Ronald S


    Transplantation of human kidney-derived cells is a potential therapeutic modality for promoting regeneration of diseased renal tissue. However, assays that determine the ability of candidate populations for renal cell therapy to undergo appropriate differentiation and morphogenesis are limited. We report here a rapid and humane assay for characterizing tubulogenic potency utilizing the well-established chorioallantoic membrane CAM) of the chick embryo. Adult human kidney-derived cells expanded in monolayer were suspended in Matrigel and grafted onto the CAM. After a week, grafts were assessed histologically. Strikingly, many of the renal cells self-organized into tubular structures. Host blood vessels penetrated and presumably fed the grafts. Immuno- and histochemical staining revealed that tubular structures were epithelial, but not blood vessels. Some of the cells both within and outside the tubules were dividing. Analysis for markers of proximal and distal renal tubules revealed that grafts contained individual cells of a proximal tubular phenotype and many tubules of distal tubule character. Our results demonstrate that the chick CAM is a useful xenograft system for screening for differentiation and morphogenesis in cells with potential use in renal regenerative medicine.

  20. A simple and sensitive flow cytometric assay for determination of the cytotoxic activity of human natural killer cells

    Radosevic, Katarina; Radosevic, K.; Garritsen, Henk S.P.; Garritsen, H.S.P.; van Graft, M.; van Graft, Marja; de Grooth, B.G.; Greve, Jan


    A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate,

  1. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.

    Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H; Xia, Menghang


    In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

  2. Comparison of frozen versus desiccated reference human red blood cells for hemagglutination assays.

    Ho, David; Schierts, Jennifer; Zimmerman, Zea; Gadsden, Isaac; Bruttig, Stephen


    Red blood cells (RBCs) are commonly used fresh or stored in frozen format for identification of patients' antibodies and serologic specificity of such antibodies at reference laboratories. However, maintaining a large pool of fresh RBCs is impossible in a blood-banking environment and blood in frozen format poses a logistic disadvantage in terms of accessibility, maintenance cost, safety, and sample recovery. This study explores an alternative, desiccation storage method for RBCs to provide a reagent that supports greater utilization and flexibility for reference laboratories. RBCs from five donors were used in the study. RBCs were processed and kept in either frozen or desiccated format. Study variables for either the frozen or the desiccated cells included cell recovery as quantified by cell counts, gross microscopic examination, and hemagglutination assays. The mean percentage of cell recovery for thawed and washed frozen RBCs was 20% versus 50% for rehydrated and washed desiccated RBCs. Microscopic examination of thawed cells from the frozen preparation showed cells with irregular shapes, a sharp contrast when compared with rehydrated cells from the desiccated preparation, where cells are mostly intact, smooth surface, and biconcave in structure. Cells in both preparations performed well in manual agglutination tests. Desiccation preservation of RBCs provides a somewhat better RBC recovery and cell structure stability, while maintaining the necessary antigen-antibody reactions for cell surface markers, which will allow desiccated RBCs to be archived in blood collecting and processing reference laboratories.

  3. Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.

    Chung, Shan; Nguyen, Van; Lin, Yuwen Linda; Kamen, Lynn; Song, An


    In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., (51)Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. A flow cytometric in vivo chalone assay using retransplanted old murine JB-1 ascites tumour cells.

    Barfod, N M


    A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G1 DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.

  5. Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay

    Mohammad Arif


    Full Text Available A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (% and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.

  6. Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens.

    Payne, J; Jones, C; Lakhani, S; Kortenkamp, A


    The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture

  7. Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

    Marie Wunsch


    Full Text Available As soon as Peripheral Blood Mononuclear Cells (PBMC are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.

  8. Effects of several salt marsh plants on mouse spleen and thymus cell proliferation using mtt assay

    Seo, Youngwan; Lee, Hee-Jung; Kim, You Ah; Youn, Hyun Joo; Lee, Burm-Jong


    In the present study, we have tested the effects of 21 salt marsh plants on cell proliferation of mouse immune cells (spleen and thymus) using MTT assay in culture. The methanolic extracts of six salt marsh plants ( Rosa rugosa, Ixeris tamagawaensis, Artemisia capillaris, Tetragonia tetragonoides, Erigeron annus, and Glehnia littoralis) showed very powerful suppressive effects of mouse immune cell death and significant activities of cell proliferation in vitro. Especially, the methanolic extract of Rosa rugosa was found to have fifteen times compared to the control treatment, demonstrating that Rosa rugosa may have a potent stimulation effect on immune cell proliferation. These results suggest that several salt marsh plants including Rosa rugosa could be useful for further study as an immunomodulating agent.

  9. Detection of DNA damage induced by heavy ion irradiation in the individual cells with comet assay

    Wada, S.; Natsuhori, M.; Ito, N.; Funayama, T.; Kobayashi, Y.


    Investigating the biological effects of high-LET heavy ion irradiation at low fluence is important to evaluate the risk of charged particles. Especially it is important to detect radiation damage induced by the precise number of heavy ions in the individual cells. Thus we studied the relationship between the number of ions traversing the cell and DNA damage produced by the ion irradiation. We applied comet assay to measure the DNA damage in the individual cells. Cells attached on the ion track detector CR-39 were irradiated with ion beams at TIARA, JAERI-Takasaki. After irradiation, the cells were stained with ethidium bromide and the opposite side of the CR-39 was etched. We observed that the heavy ions with higher LET values induced the heavier DNA damage. The result indicated that the amount of DNA damage induced by one particle increased with the LET values of the heavy ions.

  10. Aloe vera is non-toxic to cells: A microculture tetrazolium technique colorimetric assay study

    Devi Gopakumar


    Full Text Available Introduction: Aloe vera (Av, a succulent of Liliaceae family is now a widely used medicinal plant. Its′ application covers a wide spectrum of human diseases, including oral mucosa, gastric mucosa and skin. Aloe vera preparations in the form of gel, mouth washes and cream are applied topically for many oral diseases. The applications include oral lichen planus, candidiasis, oral submucous fibrosis, geographic tongue, etc. Aims and Objectives: To evaluate the cytotoxicity of Av on human fibroblasts. Materials and Methods: Aloe vera preparation (70% was applied on the fibroblast cell lineage and the cell viability was evaluated by microculture tetrazolium technique (MTT colorimetric assay. Results: The cell viability at different concentrations was measured. The cells have maintained their viability at different concentrations used in the study. Conclusion: Our study shows the cell viability at different sample concentrations of Av. This could open up wide clinical applications of Av for reactive, inflammatory and potentially malignant oral and other mucocutaneous diseases.


    Jerzy Slowinski


    Full Text Available Biological tests are efficient in reflecting the biological influences of several types of generally harmful exposures. The micronucleus assay is widely used in genotoxicity studies or studies on genomic damage in general. We present methodological aspects of cytokinesis-block micronucleus assay performed in human gliomas irradiated in vitro. Eight human glioblastoma cell lines obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany were gamma-irradiated (60Co over a dose range of 0-10 Gy. Cytokinesis-block micronucleus assay was performed to quantitate cytogenetic damage. The cells were fixed directly on dishes, stained with fluorochrome DAPI and evaluated under fluorescent and phase contrast microscope. The micronucleus frequency was expressed as a micronuclei (MN per binucleated cell (BNC ratio, calculated after scoring at least 100 BNC per dish. The frequency of spontaneous MN ranged from 0.17 to 0.613 (mean: 0.29 ± 0.14. After irradiation increase of MN frequency in the range of 0.312 - 2.241 (mean: 0.98 ± 0.68 was found at 10 Gy. Gliomas are extremely heterogenous in regard to cytogenetic effects of irradiation, as shown in this study by cytokinesis-block micronucleus assay. This test is easily performed on irradiated glioma cell lines and can assist in determining their radiosensitivity. However, in order to obtain reliable and reproducible results, precise criteria for MN scoring must be strictly followed. Simultaneous use of fluorescent and phase contrast equipment improves imaging of morphological details and can further optimize MN scoring.

  12. Quantitative assays for new families of esterified oxylipins generated by immune cells


    Phospholipid-esterified oxylipins are newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several, in particular hydro(pero)xyeicosatetraenoic acids (H(p)ETEs), hydroxyoctadecadienoic acids (HODEs), hydroxydocosahexaenoic acids (HDOHEs) and keto-eicosatetraenoic acids (KETEs), attached to...

  13. Cytotoxic Effect of Iron Oxide Nanoparticles on Mouse Embryonic Stem Cells by MTT Assay

    Homa Mohseni Kouchesfehani


    Full Text Available Background: Despite the wide range of applications, there is a serious lack of information on the impact of the nanoparticles on human health and the environment. The present study was done to determine the range of dangerous concentrations of iron oxide nanoparticle and their effects on mouse embryonic stem cells. Methods: Iron oxide nanoparticles with less than 20 nanometers diameter were encapsulated by a PEG-phospholipid. The suspension of iron oxide nanoparticles was prepared using the culture media and cell viability was determined by MTT assay. Results: MTT assay was used to examine the cytotoxicity of iron oxide nanoparticle s. Royan B1 cells were treated with medium containing different concentrations (10, 20, 30, 40, 50, and 60µg/ml of the iron oxide nanoparticle. Cell viability was determined at 12 and 24 hours after treatment which showed significant decreases when concentration and time period increased. Conclusion: The main mechanism of nanoparticles action is still unknown, but in vivo and in vitro studies in different environments suggest that they are capable of producing reactive oxygen species (ROS. Therefore, they may have an effect on the concentration of intracellular calcium, activation of transcription factors, and changes in cytokine. The results of this study show that the higher concentration and duration of treatment of cells with iron oxide nanoparticles increase the rate of cell death.

  14. Identifying tumor cell growth inhibitors by combinatorial chemistry and zebrafish assays.

    Jing Xiang

    Full Text Available Cyclin-dependent kinases (CDKs play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos. We report the successful identification of a novel lead compound that displays selective inhibitory effects on CDK2 activity, cancer cell proliferation, and tumor progression in vivo. Our approaches should have general applications in developing cell proliferation inhibitors using an efficient combinatorial chemical genetic method and integrated biological assays. The novel cell growth inhibitor we identified should have potential as a cancer therapeutic agent.

  15. EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis.

    Bandeira-Melo, Christianne; Paiva, Ligia Almeida; Amorim, Natália R T; Weller, Peter F; Bozza, Patricia T


    Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.

  16. A quantitative evaluation of cell migration by the phagokinetic track motility assay.

    Nogalski, Maciej T; Chan, Gary C T; Stevenson, Emily V; Collins-McMillen, Donna K; Yurochko, Andrew D


    Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14

  17. Assay of Peripheral Regulatory Vδ1 T Cells in Ankylosing Spondylitis and its Significance

    Wang, Hongliang; Sun, Na; Li, Ka; Tian, Jiguang; Li, Jianmin


    Background Ankylosing spondylitis (AS) involves inflammation at the sacroiliac joint and spine attachment site. This study aimed to observe the ratio and function of peripheral regulatory Vδ1 T cells in AS patients to investigate their roles in AS pathogenesis. Material/Methods Peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation from AS patients and healthy controls. Flow cytometry was used to determine the ratio between Vδ1 and CD4 T cells of PBMC in AS patients and controls. Flow cytometry sorting (FCS) was used to obtain Vδ1 and naïve CD4 T cells with purity higher than 90%. CFSE staining method was used to detect the effect of Vδ1 T cells on proliferation of naïve CD4 T cells. The effect of Vδ1 T cells on secretion of IFN-γ from naïve CD4 T cells and the ability to secrete IL-10 from Vδ1 T cells were determined by flow cytometry. Results AS patients had significantly lower Vδ1 T cell ratio in PBMC compared to controls (p<0.05), but their CD4 T cell ratio was significantly elevated (p<0.05). Functional assay showed suppression of naïve CD4 T cell proliferation and IFN-γ secretion by peripheral Vδ1 T cells in AS patients (p<0.01). AS patients also had lower IL-10 secreting level from peripheral derived Vδ1 T cells (p<0.01). Conclusions The immune suppression of peripheral Vδ1 T cell in AS patient increases the ratio of peripheral CD4 T cells and IFN-γ level, leading to AS pathogenesis. This immune suppression is mainly due to suppressed IL-10 secretion. PMID:27598263

  18. Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppression

    Auld, Douglas S.; Thorne, Natasha; Maguire, William F.; Inglese, James


    High-throughput screening (HTS) assays used in drug discovery frequently use reporter enzymes such as firefly luciferase (FLuc) as indicators of target activity. An important caveat to consider, however, is that compounds can directly affect the reporter, leading to nonspecific but highly reproducible assay signal modulation. In rare cases, this activity appears counterintuitive; for example, some FLuc inhibitors, acting through posttranslational Fluc reporter stabilization, appear to activate gene expression. Previous efforts to characterize molecules that influence luciferase activity identified a subset of 3,5-diaryl-oxadiazole-containing compounds as FLuc inhibitors. Here, we evaluate a number of compounds with this structural motif for activity against FLuc. One such compound is PTC124 {3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid}, a molecule originally identified in a cell-based FLuc assay as having nonsense codon suppression activity [Welch EM, et al., Nature (2007) 447:87–91]. We find that the potency of FLuc inhibition for the tested compounds strictly correlates with their activity in a FLuc reporter cell-based nonsense codon assay, with PTC124 emerging as the most potent FLuc inhibitor (IC50 = 7 ± 1 nM). However, these compounds, including PTC124, fail to show nonsense codon suppression activity when Renilla reniformis luciferase (RLuc) is used as a reporter and are inactive against the RLuc enzyme. This suggests that the initial discovery of PTC124 may have been biased by its direct effect on the FLuc reporter, implicating firefly luciferase as a molecular target of PTC124. Our results demonstrate the value of understanding potential interactions between reporter enzymes and chemical compounds and emphasize the importance of implementing the appropriate control assays before interpreting HTS results. PMID:19208811

  19. The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia.

    van der Hoorn, M A W G; van Luxemburg-Heijs, S A P; van Bergen, C A M; Bongaerts, R; Willemze, R; Falkenburg, J H F


    Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.

  20. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.

    Filone, Claire Marie; Heise, Mark; Doms, Robert W; Bertolotti-Ciarlet, Andrea

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by beta-galactosidase alpha-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  1. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

    Wang, Xiao; Peden, Keith; Murata, Haruhiko


    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  2. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    oxidoreductase along with minor whey constituents like osteopontin, EPV20 etc. The enterocyte migration rate is a key parameter in maintaining intestinal homeostasis and intestinal repair when recovering from infection or intestinal diseases like Crohns and ulcerative colitis. We developed a novel in vitro wound...... healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  3. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Magdalena Tary-Lehmann


    Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

  4. Adhesive-tape soft lithography for patterning mammalian cells: application to wound-healing assays.

    Shrirao, Anil B; Hussain, Ali; Cho, Cheul H; Perez-Castillejos, Raquel


    This paper introduces a benchtop method for patterning mammalian cells-i.e., for culturing cells at specific locations-on planar substrates. Compared with standard cell culture techniques, which do not allow the control of what areas of a monolayer are populated by one type of cell or another, techniques of cell patterning open new routes to cell biology. Researchers interested in cell patterning, however, are often times hindered by limited access to photolithographic capabilities. This paper shows how cells can be patterned easily with sub-millimeter precision using a non-photolithographic technique that is based on the use of office adhesive tape and poly(dimethylsiloxane) (PDMS). This method is fast (~4 h to go from a layout to have the cells patterned in the shape of such layout) and only requires materials and tools readily available in a conventional biomedical laboratory. A wound-healing assay is presented here that illustrates the potential of the technique (which we call tape-based soft lithography) for patterning mammalian cells and studying biologically significant questions such as collective cellular migration.

  5. In-channel printing-device opening assay for micropatterning multiple cells and gene analysis.

    Zhou, Hao; Zhao, Liang; Zhang, Xueji


    Herein we report an easy but versatile method for patterning different cells on a single substrate by using a microfluidic approach that allows not only spatial and temporal control of multiple microenvironments but also retrieval of specific treated cells to profile their expressed genetic information at around 10-cell resolution. By taking advantages of increased surface area of gold nanoparticles on a poly(dimethylsiloxane) (PDMS) coated substrate, cell adhesive-promotive protein, human fibronectin (hFN) can be significantly accumulated on designed regions where cells can recognize the protein and spread out. Moreover, the whole device can be easily opened by hand without any loss of patterned cells which could be retrieved by mouth-pipet. Consequently, we demonstrate the possibility of analyzing the difference of gene expression patterns between wild type MCF-7 cell and MCF/Adr (drug-resistant cell line) from less than 400 cells in total for a single comprehensive assay, including parallel experiments, controls, and multiple dose treatments. Certain genes, especially the P-glycoprotein coding gene (ABCB1), show high expression level in resistant cells compared with the wild type, suggesting a possible pathway that may contribute to the antidrug mechanism.

  6. 不同底物间接免疫荧光法检测细胞膜DNA抗体在儿童系统性红斑狼疮的临床价值研究%Indirect immunofluorescence on different cell line in detection of cell membrane DNA antibody in juvenile systemic lupus erythematosus

    茹晋丽; 靳雪琴; 吴铁鹰; 王瑞; 颉晓香; 车国柱; 李改兰; 张露月; 李小峰


    Objective To detect anti-cell membrane DNA ( cmDNA) antibody with human B lym-phocyte Raji cells and human promyelocytic leukemia HL60 cells as substrates and to compare the diagnostic value of anti-cmDNA antibody with that of anti-nucleosome antibody ( AnuA ) , anti-Sm antibody and anti-double-stranded DNA ( dsDNA) antibody in juvenile systemic lupus erythematosus ( JSLE) patients. Meth-ods We recruited 92 JSLE patients and 71 patients with other rheumatic diseases. Anti-cmDNA antibody an-dantinuclear antibody ( ANA ) was detected in patient serum by indirect immunofluorescence assays ( IIF ) . Anti-dsDNA antibody were detected by combining enzyme-linked immuno sorbent assay ( ELISA) and IIF. Anti-Sm antibody were detected by double immunodiffusion assay and immunoblotting, while anti-nucleosome antibody ( AnuA) were detected by ELISA. We collected concurrent clinical data. Results Anti-cmDNA antibody demonstrated stronger intensity of fluorescent patterns in using Raji cells as substrate than HL60 cells. JSLE patients had a significantly higher positive percentage of anti-cmDNA than patients with other rheu-matoid diseases. The sensitivity of anti-cmDNA on cell line Raji was higher than that of anti-dsDNA and anti-Sm (P0. 05) and was lower than anti-Sm and AnuA (P0. 05) and the specificity was lower than AnuA (P0.05),但低于Sm抗体及AnuA(P<0.01)。 cmDNA抗体分别与dsDNA抗体、Sm抗体及AnuA联合检测在SLE诊断中的敏感性均明显高于单独检测( P<0.05)。cmDNA抗体与SLE疾病活动度评分无相关性( P=0.907)。结论 cmDNA抗体对儿童SLE诊断的敏感性高,特异性强,可能成为儿童SLE诊断的相对特异性抗体之一。 cmDNA抗体与dsDNA抗体、Sm抗体及AnuA联合检测可提高对儿童SLE诊断的敏感性。选择Raji细胞株为底物检测cmDNA抗体较HL60细胞株更有优势。

  7. T-cell Receptor Assay and Reticulocyte-Micronuclei Assay as Biological Dosimeters for Ionizing Radiation in Humans

    Vershenya, Stanislav; Biko, Johannes; Lorenz, Reinhard; Reiners, Christoph; Stopper, Helga; Grawe, Jan; Hempel, Klaus


    In radiation accidents, biological methods are used for dosimetry if the radiation dose could not be measured by physical means. The knowledge of individual dose is a prerequisite for planning medical treatment and for health risk evaluations. In this paper we represent the summary of biodosimetrical methods used in our laboratory in the patients treated with radioiodine for thyroid cancer. The dose-response relationship was measured by the flow cytometry-based micronucleus assay in transferr...

  8. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.


    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  9. Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies.

    Belouski, Shelley S; Born, Danika; Jacques, Susan; Harder, Brandon; Reynhardt, Kai; Kaliyaperumal, Arunan; Gupta, Shalini


    We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

  10. Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish.

    Sanker, Subramaniam; Cirio, Maria Cecilia; Vollmer, Laura L; Goldberg, Natasha D; McDermott, Lee A; Hukriede, Neil A; Vogt, Andreas


    Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

  11. MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

    Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory


    A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance

  12. Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

    Valdez-Flores, Marco A; Vargas-Poussou, Rosa; Verkaart, Sjoerd; Tutakhel, Omar A Z; Valdez-Ortiz, Angel; Blanchard, Anne; Treard, Cyrielle; Hoenderop, Joost G J; Bindels, René J M; Jeleń, Sabina


    Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations. Copyright © 2016 the American Physiological Society.

  13. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal


    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

  14. Two-tiered keratinocyte assay: IL-18 production by NCTC2544 cells to determine the skin sensitizing capacity and an epidermal equivalent assay to determine sensitizer potency

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke


    method to the LLNA. Both assays are based on the use of human keratinocytes, which have been shown, over the last two decades, to play a key role in all phases of skin sensitization. First, 4 known chemicals were tested during a transferability study in which 6 laboratories participated. Three......At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it should be very important, both from ethical and economic point of view, to discriminate allergy and irritation events, and to classify sensitizers according to their potency, without...... the use of animals. The aim of the EU FP6 Integrated Project Sens-it-iv was to develop and optimize an integrated testing strategy consisting of in vitro, human cell based assays which will closely mimic sensitization mechanisms in vivo. These assays should be an alternative approach to the LLNA. The NCTC...

  15. Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016).

    Berns, Eric J; Cabezas, Maria D; Mrksich, Milan


    On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

  16. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    Simone, Giuseppina


    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence.

    Kudva, I T; Dean-Nystrom, E A


    To develop a new adherence assay, using cattle recto-anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp-2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae-independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp-2 cell adherence assay. The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto-anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food-borne pathogens that colonize cattle via adherence to this particular anatomical site. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  18. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  19. Interactive effects of metals as measured in cytotoxicity assays with established fish cell lines

    Segner, H.; Schuurmann, G. [Centre for Environmental Research, Leipzig (Germany). Dept. of Chemical Ecotoxicology


    The environmental toxicity of chemicals is often judged on the basis of toxicity tests with single compounds. One major drawback of this approach is the fact, that mixture effects occurring in aquatic ecosystems with a multitude of different chemicals are not accounted for. The present work explores the use of cytotoxicity assays with established fish cell lines as a rapid and economic approach to derive basic data on joint toxicity effects of heavy metals. For the assessment of mixture toxicity, concentration addition is taken as the reference model of no interaction, and both isobolographic analysis and calculation of mixture toxicity indices are used to analyze the effect profile of various equitoxic compound mixtures. Cytotoxic endpoints used include neutral red uptake inhibition assay as a measure of cell viability, proliferation measurements to estimate toxic effects on cell growth, and analysis of glutathion contents to estimate metabolic stress effects. The single toxicity of the metals silver, mercury, cadmium, copper, zinc, lead and nickel towards the cell lines RI from rainbow trout liver and RTG-2 from rainbow trout gonads was found to depend on the chemical softness parameter of the cations. The joint effect profile will be discussed in terms of the single effects and softness domain of the heavy metals.

  20. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  1. A large-scale (19) F MRI-based cell migration assay to optimize cell therapy.

    Bonetto, F.J.; Srinivas, M.; Weigelin, B.; Cruz, L.J.; Heerschap, A.; Friedl, P.H.A.; Figdor, C.G.; Vries, I.J.M. de


    Adoptive transfer of cells for therapeutic purposes requires efficient and precise delivery to the target organ whilst preserving cell function. Therefore, therapeutically applied cells need to migrate and integrate within their target tissues after delivery, e.g. dendritic cells (DCs) need to migra

  2. A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies

    Szeto, Y.T. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); School of Health Sciences, Macao Polytechnic Institute, Macao (China); Benzie, I.F.F. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China)]. E-mail:; Collins, A.R. [Department of Nutrition, University of Oslo, Oslo (Norway); Choi, S.W. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Cheng, C.Y. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Yow, C.M.N. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Tse, M.M.Y. [School of Nursing, Hong Kong Polytechnic University, Kowloon, Hong Kong (China)


    The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1 mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1 mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H{sub 2}O{sub 2}) increased DNA strand breaks in a dose related manner, and incubation of cells in

  3. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU


    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  4. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng


    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  5. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A


    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  6. Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells.

    Karlsson, Annika C; Martin, Jeffrey N; Younger, Sophie R; Bredt, Barry M; Epling, Lorrie; Ronquillo, Rollie; Varma, Arjun; Deeks, Steven G; McCune, Joseph M; Nixon, Douglas F; Sinclair, Elizabeth


    The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.

  7. Lack of DNA damage induced by fluoride on mouse lymphoma and human fibroblast cells by single cell gel (comet) assay

    Ribeiro,Daniel Araki; Alves de Lima, Patrícia Lepage; Marques, Mariângela Esther Alencar; Salvadori,Daisy Maria Favero


    Fluoride has widely been used in Dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury on genetic apparatus. Genotoxicity tests constitute an important part of cancer research for risk assessment of potential carcinogens. In this study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Mouse lymphoma and hum...

  8. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo


    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

  9. In vivo Comet assay--statistical analysis and power calculations of mice testicular cells.

    Hansen, Merete Kjær; Sharma, Anoop Kumar; Dybdahl, Marianne; Boberg, Julie; Kulahci, Murat


    The in vivo Comet assay is a sensitive method for evaluating DNA damage. A recurrent concern is how to analyze the data appropriately and efficiently. A popular approach is to summarize the raw data into a summary statistic prior to the statistical analysis. However, consensus on which summary statistic to use has yet to be reached. Another important consideration concerns the assessment of proper sample sizes in the design of Comet assay studies. This study aims to identify a statistic suitably summarizing the % tail DNA of mice testicular samples in Comet assay studies. A second aim is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636-97-5, CAS no. 85-28-9, CAS no. 13674-87-8, CAS no. 43100-38-5 and CAS no. 60965-26-6. Testicular cells were examined using the alkaline version of the Comet assay and the DNA damage was quantified as % tail DNA using a fully automatic scoring system. From the raw data 23 summary statistics were examined. A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells.

  10. Cytokinesis-block micronucleus assay evolves into a 'cytome' assay of chromosomal instability, mitotic dysfunction and cell death

    Fenech, Michael [CSIRO Human Nutrition, Genome Health Nutrigenomics Project, P.O. Box 10041, Adelaide BC, Adelaide, SA 5000 (Australia)]. E-mail:


    The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r = 0.75-0.77, P < 0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a 'cytome' method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations.

  11. A hard microflow cytometer using groove-generated sheath flow for multiplexed bead and cell assays.

    Thangawng, Abel L; Kim, Jason S; Golden, Joel P; Anderson, George P; Robertson, Kelly L; Low, Vyechi; Ligler, Frances S


    With a view toward developing a rugged microflow cytometer, a sheath flow system was micromachined in hard plastic (polymethylmethacrylate) for analysis of particles and cells using optical detection. Six optical fibers were incorporated into the interrogation region of the chip, in which hydrodynamic focusing narrowed the core stream to ~35 μm × 40 μm. The use of a relatively large channel at the inlet as well as in the interrogation region (375 μm × 125 μm) successfully minimized the risk of clogging. The device could withstand pressures greater than 100 psi without leaking. Assays using both coded microparticles and cells were demonstrated using the microflow cytometer. Multiplexed immunoassays detected nine different bacteria and toxins using a single mixture of coded microspheres. A549 cancer cells processed with locked nucleic acid probes were evaluated using fluorescence in situ hybridization.

  12. Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists###

    There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthe...

  13. Validation of T47D-KBluc cell assay for detection of estrogen receptor agonists and antagonists

    There is growing concern of exposure to fish, wildlife, and humans to environmental estrogens and their potential impact on reproductive health. Cell-based assays are useful tools to determine the estrogenic activity of chemicals. Confidence in in vitro assay results is strengthe...

  14. Evaluation of 1066 ToxCast Chemicals in a human stem cell assay for developmental toxicity (SOT)

    To increase the diversity of assays used to assess potential developmental toxicity, the ToxCast chemical library was screened in the Stemina devTOX quickPREDICT assay using human embryonic stem (hES) cells. A model for predicting teratogenicity was based on a training set of 23 ...

  15. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T


    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

  16. Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development.

    Carmen, Jessica; Burger, Scott R; McCaman, Michael; Rowley, Jon A


    A major challenge to commercializing cell-based therapies is developing scalable manufacturing processes while maintaining the critical quality parameters (identity, potency, purity, safety) of the final live cell product. Process development activities such as extended passaging and serum reduction/elimination can facilitate the streamlining of cell manufacturing process as long as the biological functions of the product remain intact. Best practices in process development will be dependent on cell characterization; a thorough understanding of the cell-based product. Unique biological properties associated with different types of cell-based products are discussed. Cell characterization may be used as a tool for successful process development activities, which can promote a candidate cell therapy product through clinical development and ultimately to a commercialized product.

  17. Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays.

    Blunt, Laura; Hogarth, Philip J; Kaveh, Daryan A; Webb, Paul; Villarreal-Ramos, Bernardo; Vordermeier, Hans Martin


    Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4(+) populations: CD45RO(+)CD62L(hi), CD45RO(+)CD62L(lo), CD45RO(-)CD62L(hi) and CD45RO(-)CD62L(lo) (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO(+)CD62L(hi) and CD45RO(+)CD62L(lo) phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4(+) T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4(+) T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4(+) T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO(+)CD62L(hi)).

  18. Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements.

    Basketter, David; Kolle, Susanne N; Schrage, Arnhild; Honarvar, Naveed; Gamer, Armin O; van Ravenzwaay, Bennard; Landsiedel, Robert


    The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach.

  19. A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells.

    Kawamata, Hibiki; Starkov, Anatoly A; Manfredi, Giovanni; Chinopoulos, Christos


    We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg(2+)] reported by an Mg(2+)-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg(2+). In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF(3)(-) and sodium orthovanadate (Na(3)VO(4)) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F(1)F(o)-ATPase due to its sensitivity to BeF(3)(-) and Na(3)VO(4). With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  1. A versatile assay for RNA-binding proteins in living cells.

    Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W; Castello, Alfredo


    RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

  2. Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

    Haifeng Geng

    Full Text Available Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs, inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA activity found in patients with metachromatic leukodystrophy (MLD, a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS, detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an

  3. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials.

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang


    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials.

  4. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others


    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  5. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    Sørensen, A.H.; Ahring, B.K.


    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...

  6. A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

    Jason R. Schwartz; Purvaba J. Sarvaiya; Lily E. Leiva; Maria C. Velez; Tammuella C. Singleton; Lolie C. Yu; Wayne V. Vedeckis


    Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript (exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

  7. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Uma Basavanna


    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  8. Radiometric assay of red cell and plasma cholinesterase in pesticide appliers from Minnesota.

    Potter, W T; Garry, V F; Kelly, J T; Tarone, R; Griffith, J; Nelson, R L


    In this study we demonstrate the uses of radiometric assay to detect anticholinesterases in a human population (N = 80) exposed to a broad spectrum of pesticides. The assay is nondilutional. Therefore, anticholinesterase (AChE) agents with low binding affinity can be detected. Our initial results show statistically significant exposure-related decreases in either red cell (AChE) or plasma cholinesterase activity ((butyrl)cholinesterase; BuChE) occurred not only among pesticide appliers who use organophosphates, but also among appliers of the fumigant phosphine. These data extend earlier observations made in laboratory animals exposed to this fumigant. Significant exposure-related decreases in AChE activity were seen in herbicide appliers and appear to be associated with exposure to the herbicide 2-methoxy-3,6-dichlorobenzoic acid. There was no evidence of exposure-related decreases in BuChE activity in herbicide appliers. Our in vivo data, coupled with preliminary in vitro studies of phosphine (50% AChE inhibition, 10 ppm) and 2-methoxy-3,6-chlorobenzoic acid (50% AChE and BuChE inhibition, 70 ppm), suggest that the radiometric assay may be used to detect a broader spectrum of biologically active anticholinesterase agents.

  9. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays.

    Henriksen, S T; Liu, J; Estiu, G; Oltvai, Z N; Wiest, O


    The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring, that is sufficiently accurate to justify immediate testing of the identified compounds by whole-cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of approximately 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus- and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway.

  10. Optical scatter imaging: a microscopic modality for the rapid morphological assay of living cells

    Boustany, Nada N.


    Tumors derived from epithelial cells comprise the majority of human tumors and their growth results from the accumulation of multiple mutations affecting cellular processes critical for tissue homeostasis, including cell proliferation and cell death. To understand these processes and address the complexity of cancer cell function, multiple cellular responses to different experimental conditions and specific genetic mutations must be analyzed. Fundamental to this endeavor is the development of rapid cellular assays in genetically defined cells, and in particular, the development of optical imaging methods that allow dynamic observation and real-time monitoring of cellular processes. In this context, we are developing an optical scatter imaging technology that is intended to bridge the gap between light and electron microscopy by rapidly providing morphometric information about the relative size and shape of non-spherical organelles, with sub-wavelength resolution. Our goal is to complement current microscopy techniques used to study cells in-vitro, especially in long-term time-lapse studies of living cells, where exogenous labels can be toxic, and electron microscopy will destroy the sample. The optical measurements are based on Fourier spatial filtering in a standard microscope, and could ultimately be incorporated into existing high-throughput diagnostic platforms for cancer cell research and histopathology of neoplastic tissue arrays. Using an engineered epithelial cell model of tumor formation, we are currently studying how organelle structure and function are altered by defined genetic mutations affecting the propensity for cell death and oncogenic potential, and by environmental conditions promoting tumor growth. This talk will describe our optical scatter imaging technology and present results from our studies on apoptosis, and the function of BCL-2 family proteins.



  12. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo [Nano-optoelectronics Research and Technology Laboratory (NOR.), School of Physics, Universiti Sains Malaysia, 11800, USM, Pulau Pinang (Malaysia); Mohamed, Azman Seeni; Saifuddin, Siti Nazmin [Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Pulau Pinang (Malaysia); Masudi, Sam’an Malik; Mohamad, Dasmawati [Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)


    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  13. The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay.

    Hellman, B; Vaghef, H; Boström, B


    Single cell gel electrophoresis under alkaline conditions is a technique used to detect primary DNA damage in individual mammalian cells. Cells embedded in agarose on microscope slides are subjected to lysis, unwinding of DNA and electrophoresis at high pH. After staining with a fluorescent dye, cells with DNA damage display increased migration of genetic material from the cell nucleus. The damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. The torsional moment of the tail ('tail moment') has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail. In the present paper it will be shown that the moment of inertia ('tail inertia'), a not previously described tail parameter, provides a more precise description of the distribution of individual DNA fragments within the tails. The tail inertia was also found to be the most sensitive indicator of the DNA damage induced in peripheral lymphocytes from mice given a single intraperitoneal injection of cyclophosphamide (150 mg/kg b.w.). It is concluded that the tail inertia is an important complement to other tail parameters when looking for damage of DNA with the single cell gel electrophoresis assay.

  14. Improving anticancer drug development begins with cell culture: misinformation perpetrated by the misuse of cytotoxicity assays.

    Eastman, Alan


    The high failure rate of anticancer drug discovery and development has consumed billions of dollars annually. While many explanations have been provided, I believe that misinformation arising from inappropriate cell-based screens has been completely over-looked. Most cell culture experiments are irrelevant to how drugs are subsequently administered to patients. Usually, drug development focuses on growth inhibition rather than cell killing. Drugs are selected based on continuous incubation of cells, then frequently administered to the patient as a bolus. Target identification and validation is often performed by gene suppression that inevitably mimics continuous target inhibition. Drug concentrations in vitro frequently far exceed in vivo concentrations. Studies of drug synergy are performed at sub-optimal concentrations. And the focus on a limited number of cell lines can misrepresent the potential efficacy in a patient population. The intent of this review is to encourage more appropriate experimental design and data interpretation, and to improve drug development in the area of cell-based assays. Application of these principles should greatly enhance the successful translation of novel drugs to the patient.

  15. Cell density and solvent are critical parameters affecting formazan evaluation in MTT assay

    Kellen Cristina da Silva Gasque


    Full Text Available The aim of this study was to establish the more accurate protocol for fibroblast cell viability using MTT assay. NIH/3T3 fibroblasts were seeded at the following cell densities: 3.125x10³; 1.156x10(4; 3.125x10(4; 1.156x10(5 and 3.125x10(5 cells/cm². Following 24h of seeding, MTT was added to the wells. After 4h of the MTT addition, different solvents were added to solubilize the formazan crystals: 1 HCl/SDS group- 20% SDS and 0.01 M HCl; 2 EtOH/ HAc group-50% ethanol and 1% acetic acid; 3 DMSO group- 99.5% dimethyl sulfoxide; and 4 PropOH group- 99.5% isopropanol. The absorbance values were measured using a spectrophotometer at 570 nm. The data were analyzed by 2-way ANOVA (p<0.05 and showed that the absorbance average varied according to the number of cells and solvents: HCl/SDS (0 to 0.13, EtOH/HAc (0 to 0.22, DMSO (0.76 to 1.31 and PropOH (0.66 to 1.04. The DMSO and PropOH groups presented the most appropriate protocols for NIH/3T3 fibroblasts cell viability, especially at the density of 1.156x10(4 cells/cm².

  16. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina


    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  17. BCG Infection to Human B Cells Induce Cell Apoptosis%卡介苗感染人B细胞诱导细胞凋亡

    朱旗; 冯国栋; 杨毅宁; 赵青; 王文菁; 张敏; 刘扬; 赵钢


    目的 探究卡介苗( BCG)是否能感染人B细胞及观察感染后对细胞的影响. 方法 人B细胞系Raji细胞与BCG共培养, 4、12和24h后分别用共聚焦及透射电镜观察Raji细胞对BCG的结合和摄取情况,用CCK-8法检测细胞毒性,流式An-nexin Ⅴ-PI双标法检测细胞凋亡和坏死情况. 结果 培养4、12和24h后,感染了BCG的细胞比例分别为12. 4%±2. 0%, 23. 8%±5. 1%, 25. 2%±4. 8%.BCG感染可引起细胞毒性,4h后Raji细胞存活率为75. 5%±8. 8%,12h后细胞存活率降至51. 0%± 5. 3%,24h后细胞存活率仅为21. 6%±4. 2%,而感染灭活BCG的细胞存活率均在95%以上. 进一步用流式检测凋亡坏死发现,与未感染细胞相比,Raji细胞感染BCG后凋亡,坏死均有所增加,以凋亡增加为主. 结论 BCG可以感染人B细胞并诱导细胞凋亡.%Objective To explore whether Bacillus Calmette-Guérin ( BCG) can infect human B cells and observe the effects of the infection on B cells. Methods Human B cell line Raji cells were incubated with BCG. After incubation for 4h, 12h and 24h, the direct interaction of Raji cells with BCG was observed under confocal microscopy and transmission electronic microscopy. Cytotoxity was detected by cell counting kit (CCK-8). Cell apoptosis and necrosis were assayed by flow cytometry. Results After 4h, 12h and 24h of incuba-tion, the percentage of Raji cells that were infected by BCG was 12. 4%±2. 0%, 23. 8%±5. 1%, 25. 2%±4. 8%, respectively. Live BCG infection induced cytotoxicity. The cell survival rates at 4h, 12h, 24h were 75. 5%±8. 8%, 51. 0%±5. 3%, 21. 6%±4. 2% re-spectively, while the cells infected with inactivated BCG showed a high survival rate of 95% at any time. Further analysis by flow cytome-try showed that cell apoptosis and necrosis, predominantly cell apoptosis of Raji cells were increased by BCG infection. Conclusion BCG can infect human B cells and induce cell apoptosis.

  18. Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay.

    Deman, J J; Van Larebeke, N A; Bruyneel, E A; Bracke, M E; Vermeulen, S J; Vennekens, K M; Mareel, M M


    MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

  19. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

    Cevenini, Luca; Camarda, Grazia; Michelini, Elisa; Siciliano, Giulia; Calabretta, Maria Maddalena; Bona, Roberta; Kumar, T R Santha; Cara, Andrea; Branchini, Bruce R; Fidock, David A; Roda, Aldo; Alano, Pietro


    New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.

  20. Characterization of Drug Effect on Leukemia Cells Through Single Cell Assay With Optical Tweezers and Dielectrophoresis.

    Hou, Jundi; Luo, Tao; Ng, Ka Lam; Leung, Anskar Y H; Liang, Raymond; Sun, Dong


    One of the greatest challenges in acute myeloid leukemia (AML) treatment is preventing relapse. Leukemia cells can hide in bone marrow niche or vascular niche. Hence, many chemical drugs cannot kill these cells. To characterize migration and adhesion properties of leukemia cells in specific niches, CXCR4/SDF- 1α signal pathway has been widely used for investigation. AMD3100 is treated as one of the most common chemical drugs that can inhibit this signal. In the current study, we particularly investigate the effect of AMD3100 on the adhesion property of leukemia cells on stromal cells by using engineering tools, namely, optical tweezers (OT) and dielectrophoresis (DEP), to probe single cell property. AMD3100 not only inhibits the CXCR4/SDF- 1α signal pathway but also reduces gene expression of CXCR4 and VLA-4 on leukemia cells. The drug also softens leukemia cells. This work provides a new way to investigate cell behavior under drug treatment. The use of combined engineering tools will benefit drug discovery and assessment for leukemia treatment.

  1. Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay

    Caviglia, Claudia; Zor, Kinga; Canepa, Silvia


    We investigated the combined effect of the initial cell density (12 500, 35 000, 75 000, and 100 000 cells cm−2) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing timedependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation...... between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell–substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity...... was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell...

  2. Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

    Elena Serena

    Full Text Available INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin and functional properties. The spontaneous contraction frequency was (0.83±0.2 Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2O(2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2O(2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

  3. Quantitative determination of VEGF165 in cell culture medium by aptamer sandwich based chemiluminescence assay.

    Shan, Siwen; He, Ziyi; Mao, Sifeng; Jie, Mingsha; Yi, Linglu; Lin, Jin-Ming


    In this work, we have developed a sensitive and selective chemiluminescence (CL) assay for vascular endothelial growth factor (VEGF165) quantitative detection based on two specific VEGF165 binding aptamers (Apt). VEGF is a predominant biomarker in cancer angiogenesis, and sensitive detection method of VEGF are highly demanded for both academic study and clinical diagnosis of multiple cancers. In our experiment, VEGF165 was captured in a sandwich structure assembled by two binding aptamers, one capture aptamer was immobilized on streptavidin-coated magnetic beads (MBs) and another VEGF-binding aptamer was labeled by biotin for further phosphatase conjunction. After Apt-VEGF-Apt sandwich was formed on MBs surface, alkaline phosphatase (ALP) was modified to the second aptamer to catalyze CL reaction. By applying 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) (AMPPD) as CL substrate, strong signal intensity was achieved. VEGF165 content as low as 1ng/mL was detected in standard spiked samples by our assay, and linear range of working curve was confirmed from 1 to 20ng/mL. Then our method was successfully applied for cell culture medium analysis and on-chip hypoxic HepG2-HUVEC co-culture model study with excellent accuracy equal to ELISA Kit. Our developed assay demonstrated an outstanding performance in VEGF165 quantification and may be further extended to clinical testing of important biomarkers as well as probing microchip cell culture model. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. RNA interference-mediated knockdown of brain-derived neurotrophic factor (BDNF) promotes cell cycle arrest and apoptosis in B-cell lymphoma cells.

    Xia, D; Li, W; Zhang, L; Qian, H; Yao, S; Qi, X


    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily that has been reported to be involved in a number of neurological and psychological situations. Recently, high expression level of BDNF is observed in diverse human malignancies, delineating a role of BDNF in tumorigenesis. Nevertheless, its effect on B-cell lymphoma remains unclear. In this study, RNA interference technology mediated by short hairpin RNA (shRNA) was performed to inhibit endogenous BDNF expression in B-cell lymphoma cells. Results showed that knockdown of BDNF reduced cell growth and proliferation of Raji and Ramos cells. Furthermore, down-regulation of BDNF induced a cell cycle arrest at G0/G1 phase in Raji cells, and consequently led to cell apoptosis in vitro. Meanwhile, down-regulation of Bcl-2 and up-regulation of Bax, activated caspase-3 and caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP) were observed in Raji cells when endogenous BDNF was inhibited. Besides, we also found that suppression of BDNF in Raji cells increased their sensitivity to chemotherapeutic drug, 5-Fluorouracil (5-FU). Our research provides a promising therapeutic strategy for human B-cell lymphoma by targeting BDNF.

  5. The regulation of circulating ghrelin - with recent updates from cell-based assays.

    Iwakura, Hiroshi; Kangawa, Kenji; Nakao, Kazuwa


    Ghrelin is a stomach-derived orexigenic hormone with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of appetite control in body energy homeostasis. Earlier studies revealed that circulating ghrelin levels are under the control of both acute and chronic energy status: at the acute scale, ghrelin levels are increased by fasting and decreased by feeding, whereas at the chronic scale, they are high in obese subjects and low in lean subjects. Subsequent studies revealed that nutrients, hormones, or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin secretion can assess whether and how individual factors affect ghrelin secretion from cells. In this review, on the basis of numerous human, animal, and cell-based studies, we summarize current knowledge on the regulation of circulating ghrelin levels and enumerate the factors that influence ghrelin levels.

  6. Biofilm vivacity and destruction on antimicrobial nanosurfaces assayed within a microbial fuel cell.

    Sugnaux, Marc; Fischer, Fabian


    A novel method was developed to assay the antimicrobial capacity of nanostructured surfaces for medical implants in a bicathodic microbial fuel cell. Nano-structured gold surfaces with protruding nanopillars and nanorings were investigated. Escherichia coli K12 were used as a model microbe to record electronic effects caused by the interaction with nanosurfaces. The nanostructured gold surfaces enabled power density maxima up to 1910mW/m(2), indicating fair vivacity, while flat surfaces on the nanoscale provided almost no power 0.35mW/m(2). The biofilm presence on antimicrobial nanosurfaces was confirmed by the addition of ampicillin and its bactericidal effect resulted in oscillating and declining potentiometric signals. Current density experiments showed that biofilms on antimicrobial nanostructured electrodes caused low currents, indicating that E.coli biofilm remained functional before destruction. The bicathodic microbial fuel cell sensor is a novel tool for evaluating antimicrobial effects caused by nanosurfaces and antibiotics.

  7. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M


    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  8. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU


    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  9. Radiomodifying effect of resveratrol in human rhabdomyosarcoma (RD) cell culture applying the comet assay

    Magalhaes, Vanessa D.; Rogero, Sizue O.; Vieira, Daniel P.; Okazaki, Kayo; Rogero, Jose R., E-mail: [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Cruz, Aurea S., E-mail: [Instituto Adolfo Lutz (IAL-SP), Sao Paulo, SP (Brazil)


    Cancer is considered a worldwide public health problem. Resveratrol is a defense polyphenol, synthesized naturally by a wide variety of plants according to response of ultraviolet radiation (UV) exposition or according to mechanical stress resulting of pathogens or chemical and physical agents. In vines this substance is found in elevated concentration. Thus, resveratrol is present in grape juice and wines, especially red wine. Red wines are the best dietary source of resveratrol.The protective effects performed by resveratrol during the process of cell damage, produced by oxidative effects of free radicals, are anti-inflammatory, anti-platelet and anti-carcinogenic activity, prevent or inhibit degenerative diseases, decrease incidence of cardiovascular diseases. Moreover, resveratrol is considered as a cell radioprotector. On the other hand, in some elevated concentrations resveratrol is considered as a radiosensitizing compound. The aim of this work was study in vitro the radiomodifying effect of resveratrol in human rhabdomyosarcoma (RD) cells applying the comet assay to evaluate the cellular damage and its repair capacity. In this study RD cells culture was irradiated by gamma radiation at 50 Gy and 100 Gy doses and the used resveratrol concentrations was from 15 μM to 60 μM. The protective and radioprotective effects were observed at 15 μM and 30 μM resveratrol concentrations. The resveratrol concentration of 60 μM showed cytotoxic effect to RD tumor cells and with gamma radiation presence this concentration showed no statistically significant radiosensitizing effects. (author)

  10. A rapid imageable in vivo metastasis assay for circulating tumor cells.

    Menen, Rhiana S; Pinney, Emmett; Kolostova, Katerina; Bobek, Vladimir; Suetsugu, Atsushi; Zhang, Nan; Bouvet, Michael; Hoffman, Robert M


    Circulating tumor cells (CTCs) are of great importance for cancer diagnosis, prognosis and treatment. It is necessary to improve the ability to image and analyze them for their biological properties which determine their behavior in the patient. In the present study, using immunomagnetic beads, CTCs were rapidly isolated from the circulation of mice orthotopically implanted with human PC-3 prostate cancer cells stably expressing green fluorescent protein (GFP). The PC-3-GFP CTCs were then expanded in culture in parallel with the parental PC-3-GFP cell line. Both cell types were then inoculated onto the chorioallentoic membrane (CAM) of chick embryos. Eight days later, embryos were harvested and the brains were processed for frozen sections. The IV-100 intravital laser scanning microscope enabled rapid identification of fluorescent metastatic foci within the chick embryonic brain. Inoculation of embryos with PC-3-GFP CTCs resulted in a 3 to 10-fold increase in brain metastasis when compared to those with the parental PC-3-GFP cells (passay of metastatic potential for CTCs. The chick embryo assay has future clinical application for individualizing patient therapy based on the metastatic profile of their CTCs.

  11. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    M. Anthony Moody


    Full Text Available The traditional enzyme-linked immunospot (ELISpot assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay. Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs. We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.

  12. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng


    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  13. Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.

    Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia


    The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.

  14. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    Horváth Gábor V


    Full Text Available Abstract Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

  15. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat


    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  16. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François


    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

  17. Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials

    HUANG Chun-mei; YU Lian-hui; PU Cheng-wei; WANG Xin; WANG Geng; SHEN Li-song; WANG Jian-zhong


    Background White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review.However,this last step is time consuming and requires experienced personnel.We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.Methods A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials.WBC differentials of 622 samples were also determined using both a cell counter and FCM.These results were then confirmed using manual microscopic methods.Results The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%.The resulting WBC differentials were well correlated between FCM and the cell counter (r >0.88,P <0.001),except in the case of basophils.Neutrophils,lymphocytes,and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r >0.80,P <0.001).The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%,respectively) were higher than those of the cell counter method (44.92% and 11.11%,respectively).The specificities of FCM were all above 85%,substantially better than those of the cell counter method.Conclusion These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

  18. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Jiali Xing

    Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  19. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Jertta-Riina Sarkanen


    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  20. A digital microfluidic method for multiplexed cell-based apoptosis assays.

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R


    Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.

  1. New Molecular Assay for the Proliferation Signature in Mantle Cell Lymphoma Applicable to Formalin-Fixed Paraffin-Embedded Biopsies.

    Scott, David W; Abrisqueta, Pau; Wright, George W; Slack, Graham W; Mottok, Anja; Villa, Diego; Jares, Pedro; Rauert-Wunderlich, Hilka; Royo, Cristina; Clot, Guillem; Pinyol, Magda; Boyle, Merrill; Chan, Fong Chun; Braziel, Rita M; Chan, Wing C; Weisenburger, Dennis D; Cook, James R; Greiner, Timothy C; Fu, Kai; Ott, German; Delabie, Jan; Smeland, Erlend B; Holte, Harald; Jaffe, Elaine S; Steidl, Christian; Connors, Joseph M; Gascoyne, Randy D; Rosenwald, Andreas; Staudt, Louis M; Campo, Elias; Rimsza, Lisa M


    Purpose Mantle cell lymphoma is an aggressive B-cell neoplasm that displays heterogeneous outcomes after treatment. In 2003, the Lymphoma/Leukemia Molecular Profiling Project described a powerful biomarker-the proliferation signature-using gene expression in fresh frozen material. Herein, we describe the training and validation of a new assay that measures the proliferation signature in RNA derived from routinely available formalin-fixed paraffin-embedded (FFPE) biopsies. Methods Forty-seven FFPE biopsies were used to train an assay on the NanoString platform, using microarray gene expression data of matched fresh frozen biopsies as a gold standard. The locked assay was applied to pretreatment FFPE lymph node biopsies from an independent cohort of 110 patients uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. Seventeen biopsies were tested across three laboratories to assess assay reproducibility. Results The MCL35 assay, which contained a 17-gene proliferation signature, yielded gene expression of sufficient quality to assign an assay score and risk group in 108 (98%) of 110 archival FFPE biopsies. The MCL35 assay assigned patients to high-risk (26%), standard-risk (29%), and low-risk (45%) groups, with different lengths of overall survival (OS): a median of 1.1, 2.6, and 8.6 years, respectively (log-rank for trend, P < .001). In multivariable analysis, these risk groups and the Mantle Cell Lymphoma International Prognostic Index were independently associated with OS ( P < .001 for both variables). Concordance of risk assignment across the three independent laboratories was 100%. Conclusion The newly developed and validated MCL35 assay for FFPE biopsies uses the proliferation signature to define groups of patients with significantly different OS independent of the Mantle Cell Lymphoma International Prognostic Index. Importantly, the analytic and clinical validity of this assay defines it as a reliable biomarker to

  2. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua


    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  3. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay.

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues


    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  4. In-situ gamma-ray assay of the east cell line in the 235-F Plutonium fuel form facility

    Diprete, D. [Savannah River Site (SRS), Aiken, SC (United States)


    On September 17th -19th , 2013, scientists from SRNL took a series of in-situ gamma-ray measurements in the maintenance trench beneath Cells 1-5 on the east line of the PuFF facility using a well-collimated, high-purity germanium detector. The cell interiors were assayed along with the furnaces and storage coolers that protrude beneath the cells. The detector efficiency was estimated using a combination of MCNP simulations and empirical measurements. Data analysis was performed using three gamma-rays emitted by Pu-238 (99.85 keV, 152.7 keV, and 766.4 keV) providing three independent estimates of the mass of Pu-238 holdup in each of the cells. The weighted mean of these three results was used as the best estimate of Pu-238 holdup in the East Cell Line of PuFF. The results of the assay measurements are found in the table on the following page along with the results from the scoping assay performed in 2006. All uncertainties in this table (as well as the rest of the report) are reported at 1σ. Summing the assay results and treating MDAs as M238Pu= 0 ± MDA, the total holdup in the East Cell Line was 240 ± 40 grams. This result is 100 grams lower than the previous estimate, a 0.55σ difference. The uncertainty in the Pu-238 holdup is also reduced substantially relative to the 2006 scoping assay. However, the current assay results are in agreement with the 2006 scoping assay results due to the large uncertainty associated with the 2006 scoping assays. The current assay results support the conclusion that the 2006 results bound the Pu-238 mass in Cells 1-5. These results should be considered preliminary since additional measurements of the East Cell line are scheduled for 2017 and 2018. Those measurements will provide detailed information about the distribution of Pu-238 in the cells to be used to refine the results of the current assay.

  5. LDH, proliferation curves and cell cycle analysis are the most suitable assays to identify and characterize new phytotherapeutic compounds.

    Specian, Ana Flávia L; Serpeloni, Juliana M; Tuttis, Katiuska; Ribeiro, Diego L; Cilião, Heloísa L; Varanda, Eliana A; Sannomiya, Miriam; Martinez-Lopez, Wilner; Vilegas, Wagner; Cólus, Ilce M S


    Brazilian flora biodiversity has been widely investigated to identify effective and safe phytotherapeutic compounds. Among the investigated plant species, the Byrsonima genus exhibits promising biological activities. This study aimed at evaluating the cytotoxicity of B. correifolia, B. verbascifolia, B. fagifolia and B. intermedia extracts using different assays in two cell lines (primary gastric and HepG2 cells). The different extract concentrations effects on cell viability were assayed using the MTT, aquabluer, neutral red and LDH assays. Non-cytotoxic concentrations were selected to generate cell proliferation curves and to assess cell cycle kinetics by flow cytometry. Byrsonima extracts differentially affected cell viability depending on the metabolic cellular state and the biological parameter evaluated. B. fagifolia and B. intermedia extracts exhibited lower cytotoxic effects than B. correifolia and B. verbascifolia in all assays. The results obtained with LDH and flow cytometry assays were more reliable, suggesting that they can be useful in the screening for herbal medicine and to further characterize these extracts as phytotherapeutic compounds.

  6. An assay to monitor HIV-1 protease activity for the identification of novel inhibitors in T-cells.

    Brett J Hilton

    Full Text Available The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR. The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP gene only in the presence of an effective PR Inhibitor (PI. Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.

  7. DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay.

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan


    DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.

  8. The influence of platelets, plasma and red blood cells on functional haemostatic assays.

    Bochsen, Louise; Johansson, Pär I; Kristensen, Annemarie T; Daugaard, Gedske; Ostrowski, Sisse R


    Functional whole blood haemostatic assays are used increasingly to guide transfusion therapy and monitor medical treatment and are also applied for in-vitro evaluations of the haemostatic potential of stored platelets. We investigated how the cellular and plasmatic elements, both isolated and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing plasma from platelet concentrates eliminated the TEG response and diminished the Multiplate aggregation response, but readding plasma to the pure platelet concentrates restored the response. Each of the elements in whole blood, plasma, platelets and RBC, affected the Multiplate and TEG results differently. The results emphasize that the concentrations of all cellular and plasmatic components in whole blood should be taken into account when interpreting results obtained by TEG and multiplate.

  9. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist


    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  10. A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

    Guan, Fengyi; Lu, Jiaju; Wang, Xiumei


    A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH2) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.

  11. A Comparison of the Human Buccal Cell Assay and the Pollen Abortion Assay in Assessing Genotoxicity in an Urban-Rural Gradient

    Alan da Silveira Fleck


    Full Text Available Air pollution is exacerbated near heavy traffic roads in cities. Air pollution concentration and composition vary by region and depend on urban-rural gradients. The aim of this study was to evaluate the distribution of air pollution in areas of varying population densities and to compare plant biomonitoring with an established biomarker of human exposure to traffic-related air pollution in children. The areas of study were selected near a major street in 3 different regions. Areas A, B and C represent high, intermediate and low population densities, respectively. Micronucleus assay, an established biomarker of human exposure, was performed in children from these areas. For a plant biomonitoring assay, the pollen abortion assay was performed on Bauhinia variegata in these areas. NO2 and O3 concentrations were determined by passive sampling. We report here that the pollen abortion frequency in Bauhinia variegata is correlated with NO2 concentration (P = 0.004 and is strongly associated with vehicular flow and population density in the studied areas. Micronuclei frequency in buccal cells of children was higher in the regions with more degree of urbanization (P < 0.001 following the same pattern of O3 concentrations (P = 0.030. In conclusion, our results demonstrate that high concentrations of air pollutants in Porto Alegre are related to both human and plant genotoxicity. Areas with different concentration of pollutants demonstrated to have an urbanization gradient dependent pattern which also reflected on genotoxic damage among these areas.

  12. A Comparison of the Human Buccal Cell Assay and the Pollen Abortion Assay in Assessing Genotoxicity in an Urban-Rural Gradient

    Fleck, Alan da Silveira; Vieira, Mariana; Amantéa, Sergio Luís; Rhoden, Claudia Ramos


    Air pollution is exacerbated near heavy traffic roads in cities. Air pollution concentration and composition vary by region and depend on urban-rural gradients. The aim of this study was to evaluate the distribution of air pollution in areas of varying population densities and to compare plant biomonitoring with an established biomarker of human exposure to traffic-related air pollution in children. The areas of study were selected near a major street in 3 different regions. Areas A, B and C represent high, intermediate and low population densities, respectively. Micronucleus assay, an established biomarker of human exposure, was performed in children from these areas. For a plant biomonitoring assay, the pollen abortion assay was performed on Bauhinia variegata in these areas. NO2 and O3 concentrations were determined by passive sampling. We report here that the pollen abortion frequency in Bauhinia variegata is correlated with NO2 concentration (P = 0.004) and is strongly associated with vehicular flow and population density in the studied areas. Micronuclei frequency in buccal cells of children was higher in the regions with more degree of urbanization (P < 0.001) following the same pattern of O3 concentrations (P = 0.030). In conclusion, our results demonstrate that high concentrations of air pollutants in Porto Alegre are related to both human and plant genotoxicity. Areas with different concentration of pollutants demonstrated to have an urbanization gradient dependent pattern which also reflected on genotoxic damage among these areas. PMID:25166920

  13. A comparison of the human buccal cell assay and the pollen abortion assay in assessing genotoxicity in an urban-rural gradient.

    Fleck, Alan da Silveira; Vieira, Mariana; Amantéa, Sergio Luís; Rhoden, Claudia Ramos


    Air pollution is exacerbated near heavy traffic roads in cities. Air pollution concentration and composition vary by region and depend on urban-rural gradients. The aim of this study was to evaluate the distribution of air pollution in areas of varying population densities and to compare plant biomonitoring with an established biomarker of human exposure to traffic-related air pollution in children. The areas of study were selected near a major street in 3 different regions. Areas A, B and C represent high, intermediate and low population densities, respectively. Micronucleus assay, an established biomarker of human exposure, was performed in children from these areas. For a plant biomonitoring assay, the pollen abortion assay was performed on Bauhinia variegata in these areas. NO2 and O3 concentrations were determined by passive sampling. We report here that the pollen abortion frequency in Bauhinia variegata is correlated with NO2 concentration (P = 0.004) and is strongly associated with vehicular flow and population density in the studied areas. Micronuclei frequency in buccal cells of children was higher in the regions with more degree of urbanization (P < 0.001) following the same pattern of O3 concentrations (P = 0.030). In conclusion, our results demonstrate that high concentrations of air pollutants in Porto Alegre are related to both human and plant genotoxicity. Areas with different concentration of pollutants demonstrated to have an urbanization gradient dependent pattern which also reflected on genotoxic damage among these areas.

  14. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    Kelly J Chandler

    Full Text Available The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7 and cytotoxicity (DRAQ5™/Sapphire700™ were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀ values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500 revealed significant associations for a subset of chemicals (26 that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

  15. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Wenfeng Liang

    Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

  16. Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

    Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail:; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)


    Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

  17. Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay

    Jian Su; Xiao-Sui Huang; Yi-Long Wu; Xu-Chao Zhang; She-Juan An; Wen-Zhao Zhong; Ying Huang; Shi-Liang Chen; Hong-Hong Yan; Zhi-Hong Chen; Wei-Bang Guo


    As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected forEGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cellline DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies ofEGFR, KRAS, PIK3CA, PTEN, andMEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in theHER2, NRAS, orBRAF genes. Three of the 51 mutant samples harbored double mutations: twoPIK3CA mutations coexisted withKRAS orEGFR mutations, and another KRAS mutation coexisted with aPTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay

  18. Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking

    van der Schaar, Hilde M.; Rust, Michael J.; Waarts, Barry-Lee; van der Ende-Metselaarl, Heidi; Kuhn, Richard J.; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.


    In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain SI on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by

  19. Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments.

    Kintzios, S; Bem, F; Mangana, O; Nomikou, K; Markoulatos, P; Alexandropoulos, N; Fasseas, C; Arakelyan, V; Petrou, A-L; Soukouli, K; Moschopoulou, G; Yialouris, C; Simonian, A


    The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

  20. Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes.

    Montón, Helena; Medina-Sánchez, Mariana; Soler, Joan Antoni; Chałupniak, Andrzej; Nogués, Carme; Merkoçi, Arben


    Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

  1. The influence of platelets, plasma and red blood cells on functional haemostatic assays

    Bochsen, Louise; Johansson, Pär I.; Kristensen, Annemarie Thuri


    and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet...... concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG...... and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing...

  2. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    Hokland, P; Hokland, M; Ellegaard, J


    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic...... techniques including detection of ALL antigens and terminal transferase. The malignant cells were subsequently found to be potent effectors in NK and ADCC assays. Addition of partially purified alpha-interferon to the in vitro cultures was found to have an enhancing effect on NK activity, whereas...... no modulation was seen in ADCC. These findings are discussed in the light of our present knowledge of lymphoid NK cells. Udgivelsesdato: 1981-May...

  3. Properties of kojic acid and curcumin: Assay on cell B16-F1

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir


    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  4. Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.

    Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D; Thomas, David D; Gillispie, Gregory D


    We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

  5. Use of murine embryonic stem cells in embryotoxicity assays: the embryonic stem cell test.

    Seiler, Andrea E M; Buesen, Roland; Visan, Anke; Spielmann, Horst


    The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.

  6. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar


    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

  7. Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells.

    Thakur, Ajit; Zaman, Abeyat; Hummel, Jeff; Jones, Kim; Hortelano, Gonzalo


    A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.

  8. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F., E-mail:


    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

  9. Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

    Stewart-Hutchinson, Phillip J; Szasz, Taylor P; Jaeger, Emily R; Onken, Michael D; Cooper, John A; Morley, Sharon Celeste


    Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL(-/-)) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL(-/-) B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration. © Society for Leukocyte Biology.

  10. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Bausinger, Julia; Speit, Günter


    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  11. Inter-experiment variation and dependence on culture conditions in assaying the chemosensitivity of human small cell lung cancer cell lines

    Roed, H; Christensen, I B; Vindeløv, L L


    by a logarithmic function. Even after correction for lack of proportionality the two assay systems provided significantly different dose-response curves. The stability of the chemosensitivity was tested after 25-30 weeks continuous in vitro culture or prolonged storage in liquid nitrogen. One cell line underwent...... line, dose-response curves obtained on several cell lines in different assay systems should be used in the evaluation of new drugs....

  12. In vitro pituitary and thyroid cell proliferation assays and their relevance as alternatives to animal testing.

    Jomaa, Barae; Aarts, Jac M M J G; de Haan, Laura H J; Peijnenburg, Ad A C M; Bovee, Toine F H; Murk, Albertinka J; Rietjens, Ivonne M C M


    This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

  13. A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents.

    Bergsdorf, Christian; Kropp-Goerkis, Carmen; Kaehler, Irene; Ketscher, Lars; Boemer, Ulf; Parczyk, Karsten; Bader, Benjamin


    Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio

  14. Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

    Frick A


    were generated using GraphPad Prism 6. Results: Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion: Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies. Keywords: immunomodulation, cytotoxicity, chemotherapy, precision medicine

  15. Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

    Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro


    Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP ). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

  16. Multiplex assay for live-cell monitoring of cellular fates of amyloid-β precursor protein (APP.

    Maria Merezhko

    Full Text Available Amyloid-β precursor protein (APP plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments.

  17. Cell-Free Expression of Protein Kinase A for Rapid Activity Assays

    Donna M. Leippe


    Full Text Available Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.

  18. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido


    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

  19. Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products.

    Randlev, Britta; Huang, Li-chun; Watatsu, Mitsuko; Marcus, Matthew; Lin, Andy; Shih, Shian-Jiun


    GVAX immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

  20. Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin.

    Poletta, G L; Gigena, F; Loteste, A; Parma, M J; Kleinsorge, E C; Simoniello, M F


    Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68±5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H2O2, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150μg/l (DI: 239.62±6.21) and 0.300μg/l (270.63±2.09) compared to control (150.25±4.38) but no effect was observed at 0.075μg/l (168.50±10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H2O2), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.

  1. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Ricardo Carneiro Borra


    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  2. Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

    Manuel A F V Gonçalves

    Full Text Available Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and

  3. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi


    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  4. Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells.

    Bindra, Ranjit S; Goglia, Alexander G; Jasin, Maria; Powell, Simon N


    Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

  5. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John


    Dipeptidyl peptidase 1 (DPP1) (EC; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  6. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

    Peper, Janet Kerstin; Schuster, Heiko; Löffler, Markus W; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Stevanović, Stefan


    The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

  7. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Resende, Flávia A; de Oliveira, Ana Paula S; de Camargo, Mariana S; Vilegas, Wagner; Varanda, Eliana A


    Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA) and the MCF-7 proliferation assay (E-screen), since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  8. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Flávia A Resende

    Full Text Available Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA and the MCF-7 proliferation assay (E-screen, since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  9. Human B cells have an active phagocytic capability and undergo immune activation upon phagocytosis of Mycobacterium tuberculosis.

    Zhu, Qi; Zhang, Min; Shi, Ming; Liu, Yang; Zhao, Qing; Wang, Wenjing; Zhang, Guangyun; Yang, Longxiu; Zhi, Jin; Zhang, Lin; Hu, Gengyao; Chen, Pin; Yang, Yining; Dai, Wen; Liu, Tingting; He, Ying; Feng, Guodong; Zhao, Gang


    The paradigm that B cells are nonphagocytic was taken for granted for a long time until phagocytic B cells were found in early vertebrate animals. Thereafter, limited evidence has shown that human B cells may also internalize bacteria. However, whether human B cells can actively phagocytose bacteria has been less extensively investigated; in particular, the mechanisms and significance of the phagocytosis require clarification. Here, we show that the human Raji B cell line can phagocytose both live and dead Mycobacterium tuberculosis (Mtb), and the phagocytosed Mtb in turn affects the immune functions of the B cells. After incubation of Raji cells with Mtb, our confocal microscopy, electron microscopy and flow cytometry data showed that Raji cells effectively engulfed Mtb as well as latex beads. The phagocytic rate was proportional to the incubation time and the amount of Mtb or beads added. Additionally, we found that normal human serum could enhance the ability of Raji cells to phagocytose Mtb, while heat-inactivated serum reversed this promoting effect. The phagocytic process of B cells could partially be inhibited by cytochalasin B, an actin inhibitor. Importantly, the phagocytosed Mtb could regulate B cell immune functions, such as stimulating IgM production and upregulating the expression of the antigen-presenting costimulatory molecules CD80 and CD86. Therefore, our results provide the first evidence that human B cells can phagocytose Mtb in an active manner that is independent of bacterial viability, and phagocytosed Mtb can in turn regulate the immune activation of B cells.

  10. Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay.

    Sánchez-Fortún, S; Llorente, M T; Castaño, A


    A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluorescence reader at room temperature for less than 1h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC(50(48)) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC(50(48)) value, and the results obtained indicate a strain scission factor (SSF) of 0.126+/-0.014, 0.181+/-0.014, 0.217+/-0.013, and 0.245+/-0.013 in cell suspensions, and 0.077+/-0.019, 0.107+/-0.014, 0.151+/-0.014, and 0.202+/-0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023+/-0.009, 0.033+/-0.017, 0.068+/-0.012, and 0.088+/-0.015 in cell suspensions, and 0.033+/-0.010, 0.044+/-0.011, 0.080+/-0.009, and 0.093+/-0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2(1/100 IC(50(48))) and 300(1/10 IC(50(48))) Hg/L of tetrakis

  11. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    Gottfried H. Kellermann


    Full Text Available Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™ to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

  12. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.


    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  13. Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

    Westerink, W.M.; Schirris, T.J.J.; Horbach, G.J.; Schoonen, W.G.


    In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these

  14. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Julio Alonso-Padilla


    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  15. Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

    Roberta Fedriga


    Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

  16. Cell size matters in gamma-H2AX assay for low-dose alpha particle effect assessment

    Lee, Ui seob; Kim, Eun Hee [Seoul National University, Daejeon (Korea, Republic of)


    Gamma-H2AX assay is an immuno-fluorescence experiment that enables detecting the location and number of DNA double strand breaks (DSBs) in cells. Under uniform radiation beam intensity, cells would respond with similar numbers of gamma-H2AX if they are similar in cross section. If cells are not represented by a common size, however, a larger cell has a greater chance of radiation exposure and has a better chance of counting a greater number of foci. In other words, the cell size distribution would be reflected in the FPC distribution. In the conventional gamma-H2AX assay, the mean FPC value solely indicates the level of cellular damage under a certain radiation exposure. The purpose of this study is to investigate the FPC distribution in connection with the cell size distribution. The high-LET alpha beam was employed for radiation exposure so that a single track of radiation leaves a meaningful amount of energy in the cell. Gamma-H2AX is a powerful tool for investigating the cellular response at low-dose exposure. If the gamma-H2AX assay is performed with cells of the same size, 'the average number of foci per cell' may accord with the overall response of sample cells to radiation exposure. With cells of non-uniform size, however, one should be cautious in taking the value as an index of the severity in cellular effect of radiation exposure. According to our experiments, a portion of sample cells carried DSBs of more than 5 times greater number than the mean FPC value and might play a critical role in radio-response.

  17. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard


    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids.

  18. Improvement of the BALB/c-3T3 cell transformation assay: a tool for investigating cancer mechanisms and therapies.

    Poburski, Doerte; Thierbach, René


    The identification of cancer preventive or therapeutic substances as well as carcinogenic risk assessment of chemicals is nowadays mostly dependent on animal studies. In vitro cell transformation assays mimic different stages of the in vivo neoplastic process and represent an excellent alternative to study carcinogenesis and therapeutic options. In the BALB/c-3T3 two-stage transformation assay cells are chemically transformed by treatment with MCA and TPA, along with the final Giemsa staining of morphological aberrant foci. In addition to the standard method we can show, that it is possible to apply other chemicals in parallel to identify potential preventive or therapeutic substances during the transformation process. Furthermore, we successfully combined the BALB/c cell transformation assay with several endpoint applications for protein analysis (immunoblot, subcellular fractionation and immunofluorescence) or energy parameter measurements (glucose and oxygen consumption) to elucidate cancer mechanisms in more detail. In our opinion the BALB/c cell transformation assay proves to be an excellent model to investigate alterations in key proteins or energy parameters during the different stages of transformation as well as therapeutic substances and their mode of action.

  19. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.


    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  20. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the

  1. Measurement of oxidatively-induced clustered DNA lesions using a novel adaptation of single cell gel electrophoresis (comet assay).

    Georgakilas, Alexandros G; Holt, Stewart M; Hair, Jessica M; Loftin, Charles W


    The two basic groups of complex DNA damage are double-strand breaks (DSBs) and non-DSB oxidatively-induced clustered DNA lesions (OCDLs). The single-cell gel electrophoresis (SCGE) or comet assay has been widely used for the detection of low levels of various types of DNA lesions including single-strand breaks (SSBs), DSBs, and oxidized bases per individual cell. There are limited data on the use of the comet assay for the detection of non-DSB clustered DNA lesions using different repair enzymes as enzymatic probes. This unit discusses a novel adaptation of the comet assay used to measure these unique types of lesions. Until now OCDL yields have been measured using primarily pulsed-field agarose gel electrophoresis. The advantages offered by the current approach are: (1) measurement of OCDL levels per individual cell; (2) use of a small number of cells (∼10,000) and relatively low doses of ionizing radiation (1 to 2 Gy) or low levels of oxidative stress, which are not compatible with standard agarose gel electrophoresis; and finally, (3) the assay is fast and allows direct comparison with pulsed-field gel electrophoresis results.

  2. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.


    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  3. Estrogenic activity of estradiol and its metabolites in the ER-CALUX assay with human T47D breast cells

    Hoogenboom, L.A.P.; Haan, de L.; Hooijerink, D.; Bor, G.; Murk, A.J.; Brouwer, A.


    A number of metabolites of 17β-estradiol were tested for their estrogenic activity using the ER-CA-LUX assay based on the increased expression of luciferase in exposed T47D breast cancer cells. E2β and estrone showed similar potencies in the test, whereas E2α was 100 times less active. Incubation of


    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. WilsonU.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  5. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph


    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  6. High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay

    Robert W. Yucha


    Full Text Available Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent infection. We therefore developed a microfluidic single-cell-in-droplet (scdPCR assay to directly measure the number of CD4+ T cells that produce unspliced (usRNA and multiply spliced (msRNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin or T cell receptor (TCR stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4+ T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous—increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies.

  7. The Application of a Highly Purified Rat Leydig Cell Assay as a Complement to the H295R Steroidogenesis Assay for the Evaluation of Toxicant Induced Alterations in Testosterone Production

    U.S. Environmental Protection Agency — The greater dynamic range of testosterone production in a highly purified rat Leydig cell assay permitted the detection of chemical induced inhibition that was not...

  8. Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure

    Schulpen, Sjors H. W.; Theunissen, Peter T.; Pennings, Jeroen L. A.; Piersma, Aldert H.


    Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural EST

  9. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    Barington, T; Heilmann, C


    SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti...

  10. Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay

    de Jong, Steven; Zijlstra, J G; Timmer-Bosscha, H; Mulder, N H; de Vries, Liesbeth


    Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under

  11. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    DiDone, Louis; Oga, Duana; Krysan, Damian J


    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. Copyright © 2011 John Wiley & Sons, Ltd.

  12. The statistical performance of an MCF-7 cell culture assay evaluated using generalized linear mixed models and a score test.

    Rey deCastro, B; Neuberg, Donna


    Biological assays often utilize experimental designs where observations are replicated at multiple levels, and where each level represents a separate component of the assay's overall variance. Statistical analysis of such data usually ignores these design effects, whereas more sophisticated methods would improve the statistical power of assays. This report evaluates the statistical performance of an in vitro MCF-7 cell proliferation assay (E-SCREEN) by identifying the optimal generalized linear mixed model (GLMM) that accurately represents the assay's experimental design and variance components. Our statistical assessment found that 17beta-oestradiol cell culture assay data were best modelled with a GLMM configured with a reciprocal link function, a gamma error distribution, and three sources of design variation: plate-to-plate; well-to-well, and the interaction between plate-to-plate variation and dose. The gamma-distributed random error of the assay was estimated to have a coefficient of variation (COV) = 3.2 per cent, and a variance component score test described by X. Lin found that each of the three variance components were statistically significant. The optimal GLMM also confirmed the estrogenicity of five weakly oestrogenic polychlorinated biphenyls (PCBs 17, 49, 66, 74, and 128). Based on information criteria, the optimal gamma GLMM consistently out-performed equivalent naive normal and log-normal linear models, both with and without random effects terms. Because the gamma GLMM was by far the best model on conceptual and empirical grounds, and requires only trivially more effort to use, we encourage its use and suggest that naive models be avoided when possible. Copyright 2006 John Wiley & Sons, Ltd.

  13. Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens.

    Albino, A P; Huang, X; Jorgensen, E; Yang, J; Gietl, D; Traganos, F; Darzynkiewicz, Z


    DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 microg/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G(1) vs S vs G(2)/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.

  14. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    Walter, M.N.M. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom); Wright, K.T.; Fuller, H.R. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); MacNeil, S. [Kroto Research Institute and Centre for Nanoscience and Technology, Sheffield University, Sheffield, S1 2UE (United Kingdom); Johnson, W.E.B., E-mail: [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)


    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  15. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol.

    Schulpen, Sjors H W; de Jong, Esther; de la Fonteyne, Liset J J; de Klerk, Arja; Piersma, Aldert H


    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as βIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of βIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.

  16. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji


    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

  17. Comparative tumor promotion assessment of e-cigarette and cigarettes using the in vitro Bhas 42 cell transformation assay.

    Breheny, Damien; Oke, Oluwatobiloba; Pant, Kamala; Gaça, Marianna


    In vitro cell transformation assays (CTA) are used to assess the carcinogenic potential of chemicals and complex mixtures and can detect nongenotoxic as well as genotoxic carcinogens. The Bhas 42 CTA has been developed with both initiation and promotion protocols to distinguish between these two carcinogen classes. Cigarette smoke is known to be carcinogenic and is positive in in vitro genotoxicity assays. Cigarette smoke also contains nongenotoxic carcinogens and is a tumour promoter and cocarcinogen in vivo. We have combined a suite of in vitro assays to compare the relative biological effects of new categories of tobacco and nicotine products with traditional cigarettes. The Bhas promotion assay has been included in this test battery to provide an in vitro surrogate for detecting tumor promoters. The activity of an electronic cigarette (e-cigarette; Vype ePen) was compared to that of a reference cigarette (3R4F) in the promotion assay, using total particulate matter (TPM)/aerosol collected matter (ACM) and aqueous extracts (AqE) of product aerosol emissions. 3R4F TPM was positive in this assay at concentrations ≥6 µg/mL, while e-cigarette ACM did not have any promoter activity. AqE was found to be a lesssuitable test matrix in this assay due to high cytotoxicity. This is the first study to use the Bhas assay to compare tobacco and nicotine products and demonstrates the potential for its future application as part of a product assessment framework. These data add to growing evidence suggesting that e-cigarettes may provide a safer alternative to traditional cigarettes. Environ. Mol. Mutagen. 58:190-198, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

    Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie


    We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

  19. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    Zhu, Xiaoming; Fu, Afu [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Luo, Kathy Qian, E-mail: [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)


    Highlights: Black-Right-Pointing-Pointer An endothelial cell apoptosis assay using FRET-based biosensor was developed. Black-Right-Pointing-Pointer The fluorescence of the cells changed from green to blue during apoptosis. Black-Right-Pointing-Pointer This method was developed into a high-throughput assay in 96-well plates. Black-Right-Pointing-Pointer This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z Prime factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  20. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C


    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.

  1. Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants.

    Nyffeler, Johanna; Karreman, Christiaan; Leisner, Heidrun; Kim, Yong Jun; Lee, Gabsang; Waldmann, Tanja; Leist, Marcel


    Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.

  2. Induction of epstein-barr virus (EBV lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

    benmansour Riadh


    Full Text Available Abstract Background We investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation. Results After 48 h (peak of lytic cycle, a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively. Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively. Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively. DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls. Conclusion The results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV.

  3. A sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells.

    Lijian Shao

    Full Text Available Hematopoietic stem cells (HSCs are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs. Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34(+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34(+CD38(- cells are less proficient in the repair of DSBs by NHEJ than HPCs (CD34(+CD38(+ cells. In contrast, mouse quiescent HSCs (Pyronin-Y(low LKS(+ cells and cycling HSCs (Pyronin-Y(hi LKS(+ cells repaired the damage more efficiently than HPCs (LKS(- cells. The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.

  4. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J


    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  5. Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

    WANG Jin-e; WANG Cheng-ke; LIU Dian-jun; WANG Zhen-xin


    The interactions of antibiotic with living cells were studied by iectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.

  6. Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells.

    Shenoy, Niraj; Stenson, Mary; Lawson, Joshua; Abeykoon, Jithma; Patnaik, Mrinal; Wu, Xiaosheng; Witzig, Thomas


    Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.Laboratory Investigation advance online publication, 27 February 2017; doi:10.1038/labinvest.2017.18.

  7. TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

    C.A. Dalla Torre


    Full Text Available Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

  8. Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

    Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard;


    The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators...

  9. Use of ImageJ to recover information from individual cells in a G protein-coupled receptor assay.

    Trabuco, João R C; Martins, Sofia Aires M; Prazeres, Duarte Miguel F


    Live-cell assays used in GPCR research often rely on fluorescence techniques that generate large amounts of raw image data. Consequently, the capacity to accurately and timely extract useful information from image and video data has become more and more important. Image J is an open-source program that provides powerful tools with a simple interface designed to fit the needs of image analysis of most researchers. In this chapter, Image J routines to extract information from individual cells in a calcium GPCR assay are described. In these routines, individual cells in the same image/video data can be separated using either a progressive threshold or a local threshold method. Both methods can be optimized to either a maximum number of selection or maximum area selected resulting in conceptually distinct selections.

  10. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

    Fu, Ying; Lu, Danqin; Lin, Bin; Sun, Qianqian; Liu, Kai; Xu, Lili; Zhang, Shengping; Hu, Chen; Wang, Chuangui; Xu, Zhiai; Zhang, Wen


    Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

  11. Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells.

    Zór, Kinga; Mark, Christina; Heiskanen, Arto; Madsen, Claus Krogh; Dufva, Martin; Emnéus, Jenny; Brinch-Pedersen, Henrik; Finnie, Christine


    The barley aleurone layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the aleurone layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same aleurone layer samples over time, in the presence or absence of plant hormones and other effectors. The system is also amenable to transient gene expression by particle bombardment, with simultaneous monitoring of cell death. In conclusion, the easy to handle and efficient experimental setup developed here enables continuous monitoring of tissue samples, parallelisation of assays and single cell analysis, with potential for time course studies using any plant tissue that can be immobilised, for example leaves or epidermal peels. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Detection of bladder transitional cell carcinoma: urinary hTERT assay versus urine cytology

    Yahyazadeh SR


    Full Text Available "nBackground: Transitional Cell Carcinoma (TCC of bladder is the second most common urogenital malignancy and because of its high rate of recurrence (two third of tumors recur vigilant surveillance is necessary. There have been a lot of efforts to find a proper biomarker for detecting urothelial cancers because available methods are expensive and invasive (like cystoscopy or have a low degree of sensitivity (like urine cytology. Urothelial malignancies, like other cancers tend to express a large amount of telomerase. The aim of this study was to evaluate the possible application of voided urine human telomerase reverse transcriptase (hTERT mRNA assay in detecting low-grade bladder carcinoma in comparison with urine cytology. "nMethods: Voided urine samples were collected from 49 patients who were supposed to go under operation. Samples were examined by both Quantitative Real-time RT-PCR (for measuring hTERT mRNA level and cytology; the results were then compared to the final pathologic studies. "nResults: Regardless of clinical stage and or pathological grade of tumor, sensitivity of telomerase test and urine cytology was 74% and 16% respectively. There was a strong correlation between results of urine cytology and stage and/or grade of tumor; however, sensitivity of telomerase test was acceptable regardless of stage and or grade of tumor. There was a statistically significant difference between sensitivity of urine cytology and telomerase test (p<0.001. "nConclusion: Detection of hTERT-mRNA can potentially be used as a non-invasive method for diagnosis and follow up of bladder carcinoma instead of urine cytology.

  13. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    McGee Dennis


    Full Text Available Abstract Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96 and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%. DOX-96 showed a lower limit of detection (1.25 × 10(4 cells/ml; whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4 cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring.

  14. Impedance spectroscopy applied to the fast wounding dynamics of an electrical wound-healing assay in mammalian cells

    Bellotti, Mariela I.; Giana, Fabián E.; Bonetto, Fabián J.


    Electrical wound-healing assays are often used as a means to study in vitro cell migration and proliferation. In such analysis, a cell monolayer that sits on a small electrode is electrically wounded and its spectral impedance is then continuously measured in order to monitor the healing process. The relatively slow dynamics of the cell healing have been extensively studied, while those of the much faster wounding phase have not yet been investigated. An analysis of the electrical properties of a particular cell type during this phase could give extra information about the changes in the cell membrane due to the application of the wounding current, and could also be useful to optimize the wounding regime for different cell types. The main issue when trying to register information about these dynamics is that the traditional measurement scheme employed in typical wound-healing assays doesn’t allow the simultaneous application of the wounding signal and measurement of the system’s impedance. In this paper, we overcome this limitation by implementing a measurement strategy consisting of cycles of fast alternating low- and high-voltage signals applied on electrodes covered with mammalian cells. This approach is capable of registering the fast impedance changes during the transient regime corresponding to the cell wounding process. Furthermore, these quasi-simultaneous high- and low-voltage measurements can be compared in order to obtain an empirical correlation between both quantities.

  15. Cytotoxicity of PEGylated graphene oxide on lymphoma cells.

    Du, Li; Wu, Shaoling; Li, Yanhui; Zhao, Xindong; Ju, Xiaoyan; Wang, Yuzhen


    Graphene oxide (GO) is a hotspot, especially in the field of biomedical. However, the clinical application of GO is still faces a lot of challenges. In order to improve the solubility and biocompatibility of GO, polyethylene glycol (PEG) was grafted on the surface of graphene oxide by amide reaction. PEGylated graphene oxide (PEG-GO) was characterized using Fourier transform infrared spectroscopy (FTIR). The stability of PEG-GO detected in different solutions. Raji cell was selected as a lymphoma cell model to study the cytotoxicity of PEG-GO. Cell viability was detected using the Cell Counting Kit-8 assay. Cells were treated with different concentrations (10-100 μg/mL) of PEG-GO at different time points (6, 12, and 24 h). The FTIR spectrum of PEG-GO indicated that polyethylene glycol was successfully grafted onto GO. PEG-GO had excellent stability in all solutions. Cells treated with PEG-GO (10-100 μg/mL) for 24 hours had survival rates were over 80%. These results demonstrate that PEG-GO had an excellent dispersion in biological solutions and the toxicity of PEG-GO to lymphoma cells was low. The paper may provide cytological evidence for the application of PEG-GO in medicine.

  16. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M


    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  17. Cell-based screening assay for anti-inflammatory activity of bioactive compounds

    Meijer, Kees; Vonk, Roel J.; Priebe, Marion G.; Roelofsen, Han


    Excess dietary intake may induce metabolic inflammation which is associated with insulin resistance and cardiovascular disease. Recent evidence indicates that dietary bioactive compounds may diminish metabolic inflammation. To identify anti-inflammatory bioactives, we developed a screening assay

  18. The application of single cell gel electrophoresis or comet assay to human monitoring studies Aplicacion de la electroforesis unicelular o ensayo cometa en estudios de monitoreo humano


    Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a g...

  19. Caspase-3-dependent Cell Death in B lymphocyte Caused by Pseudomonas aeruginosa Pyocyanin

    Heni Susilowati


    Full Text Available Objective: The aim of this study was to investigate cellular responses of B lymphocyte to the exposure of pyocyanin and the role of caspase-3 in its molecular mechanism. Methods: B lymphocytes (Raji cells were cultured overnight prior to the experiments. Cell culture in five replications were then exposed to various concentrations of pyocyanin and incubated for 24 h in antibiotics-free medium. MTT assay was performed to analyze the cytotoxicity effect of pyocyanin. In separated experiments, the cells were cultured with pyocyanin and addressed for cell morphological a