3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium and thiazolyl-blue

The species of the genus Acanthamoeba are opportunistic protozoan parasites that cause different diseases in humans, such as amoebic keratitis and granulomatous encephalitis. The rise in the rate of Acanthamoeba keratitis, mainly due to the increase in contact lens wearers, turns the development of viability assays using a multi-well plate reader as a tool for screening new antiamoebic agents in vitro into an important goal. In our study, the viability assays PrestoBlue®, resazurin sodium salt, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and CellTiter96® were tested for their suitability as time-saving alternatives to the classical manual or direct-counting method, assessing the effect of the antiamoebic agent chlorhexidine digluconate and temperature on Acanthamoeba castellanii (ATCC® 30234™) and Acanthamoeba polyphaga 2961. Although resazurin and MTT have already been previously used in amoeba viability assays to test the activities of antiamoebic agents in vitro, it is the first time that PrestoBlue® and CellTiter96® are used for this purpose. Results indicated that the viability assays were strain-dependent leading in some cases to an overestimation of the real situation of viable cells. This implies that each viability assay ought to be set up for each amoeba strain studied.

Topics: Acanthamoeba; Acanthamoeba castellanii; Acanthamoeba Keratitis; Antiprotozoal Agents; Chlorhexidine; Contact Lenses; Humans; Indicators and Reagents; Oxazines; Tetrazolium Salts; Thiazoles; Trophozoites; Xanthenes

The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC(50) concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature.. We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC(50) concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays.. These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.

Topics: Anticarcinogenic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coloring Agents; Female; Flavonoids; Humans; Inhibitory Concentration 50; Male; Phenols; Polyphenols; Reagent Kits, Diagnostic; Tea; Tetrazolium Salts; Thiazoles

An in vitro reproductive cell-based toxicity assay was developed using MLTC-1 (murine Leydig tumour cell line) in order to examine the reproductive toxicity of two novel nanopharmaceutical compounds, namely ethylene glycol mono allyl ether and poly(ethylene glycol) octa-functionalized polyhedral oligomeric silsesquioxane. Three commonly used cytotoxicity assays, namely the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and Crystal Violet assays, were compared, and the MTT assay proved to be the most accurate and reproducible for the MLTC-1 cell line. The doubling rate of the MLTC-1 cells was 30+/-3.5 h and the optimal seeding density for the MTT assay was 20000 cells per well, and the optimized MTT assay utilized a 4 h cell adherence followed by incubation with 0.5 mg/ml MTT for 1 h. The intra- and inter-assay CV (coefficient of variation) values were 12.3 and 11% respectively. MLTC-1 cells only produce the reproductive hormone progesterone in response to hCG (human chorionic gonadotropin), which stimulated progesterone production dose-dependently from 0 to 100 m.i.u. (milliinternational units)/ml (2706+/-1118 ng/ml). H(2)O(2) as a negative control killed 100% of cells at 1000 microg/ml. The two nanopharmaceutical compounds were cytotoxic at concentrations > or =0.1 microg/ml, but hCG decreased cytotoxicity to > or =1000 microg/ml (P<0.001). hCG-stimulated progesterone synthesis afforded some protection against the cytotoxic effects of the two novel nanotechnology compounds; therefore doses < or =100 microg/ml and an exposure period of 1 h would be recommended for testing in in vivo animal reproductive assays.

Topics: Animals; Cell Count; Cell Line, Tumor; Cell Proliferation; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ethylene Glycol; Gentian Violet; Male; Mice; Mice, Inbred C57BL; Molecular Structure; Organosilicon Compounds; Progesterone; Radioimmunoassay; Reproducibility of Results; Tetrazolium Salts; Thiazoles; Toxicity Tests

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment of cell proliferation over 14 day culture.

Topics: Animals; Biological Assay; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Chondrocytes; Colorimetry; DNA; Fibrocartilage; Mitochondria; Sensitivity and Specificity; Sheep; Tetrazolium Salts; Thiazoles

The sensitivity of [3H]thymidine incorporation and MTT/MTS colorimetric bioassays for detection and quantitation of murine and human IL-4 and IL-2 were compared in CT.4S, CT.h4S and HT-2 bioassays respectively. We reasoned that low levels of cytokine, insufficient to induce cell proliferation (thus, DNA synthesis and [3H]thymidine incorporation), may be sufficient to maintain the viability of the bioassay cells in culture. Because colorimetric assays such as those employing MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfonyl)-2H- tetrazolium) measure conversion of these salts to intensely colored formazan products by mitochondrial enzymes independent of whether proliferation is induced, we reasoned that such assays could be superior for detection of low levels of cytokine protein. Direct comparison of these approaches demonstrated that the MTT- and MTS-based assays were consistently able to detect 2-16-fold lower cytokine levels than methods based on [3H]thymidine incorporation. Moreover, the MTT and MTS assays exhibited higher precision with standard deviations of < 1-4% vs. 5-15% for thymidine incorporation. This finding is of particular importance in approaches such as limiting dilution analysis, or primary bulk culture of antigen-stimulated human lymphocytes, where levels of cytokine production may be extremely low.

Topics: Animals; Biological Assay; Cell Line; Colorimetry; Humans; Interleukin-2; Interleukin-4; Lymphocytes; Mice; Sensitivity and Specificity; Tetrazolium Salts; Thiazoles; Thymidine; Tritium

Topics: Allopurinol; Amyloid beta-Peptides; Animals; Antimycin A; Antineoplastic Agents; Cisplatin; Coloring Agents; Deoxyglucose; Dicumarol; Doxorubicin; Humans; Mitochondria; Oxazines; Oxidation-Reduction; Oxygen Consumption; PC12 Cells; Peptide Fragments; Rats; Rotenone; Tetrazolium Salts; Thiazoles; Xanthenes

A colorimetric end-point dilution assay was developed for the titration of rotavirus-containing samples that uses commercially available tetrazolium dyes as an indicator of virus infection. This assay offers several advantages over both plaque assays and traditional end-point dilution methods. The latter assays require manual counting of plaques or the scoring of wells for the presence of virus based on observed cytopathic effects. The colorimetric end-point dilution assay enables the scoring of wells based upon absorbance readings alone, thereby eliminating time-consuming and subjective manual screenings. This method also has the potential for automating the analysis of large numbers of samples. Virus titers of human-bovine rotavirus reassortants obtained using this method are comparable to those determined by plaque assay. The scoring of wells based on absorbance readings was also found to agree with manual scoring of cytopathic effects and with the production of viral antigen.

Topics: Animals; Antigens, Viral; Capsid; Capsid Proteins; Cattle; Cell Line; Chlorocebus aethiops; Colorimetry; Coloring Agents; Cytopathogenic Effect, Viral; Humans; Macaca mulatta; Reassortant Viruses; Rotavirus; Tetrazolium Salts; Thiazoles; Titrimetry; Vero Cells

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.

Topics: Animals; Cell Count; Cell Division; Cells, Cultured; Coloring Agents; Drug Evaluation; Drug Screening Assays, Antitumor; Formazans; Humans; Methylphenazonium Methosulfate; Mice; Spectrophotometry; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured