Cytotoxicity assay methods

Niles, A., Moravec, R., and Riss, T.L. 2008. Multiplex caspase activity and cytotoxicity assays. Methods Mol. Biol. 414, 151-162. 

Wlodkowic, D. Faley, S. Darzynkiewicz, Z. Cooper, J. M. Real-time cytotoxicity assays. Methods Mol. Biol. 2011, 731, 285-291. 

H. Gazzano-Santoro, P. Ralph, T. Ryskamp, A. Chen, and V. Mukku, A non-radioactive complement-depen-dent cytotoxicity assay for anti-CD20 monoclonal antibody, J. Imunol. Methods, 202, 163 (1997). 

Fischer, K. and Mackensen, A., The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity, Methods, 31, 135, 2003. 

The test material, test cells, used, method of treatment, harvesting of cells, cytotoxicity assay, and so on, should be clearly stated as well as the statistical methods used. Richardson et al. (1989) recommend that comparison be made between the frequencies in control cells and at each dose level using Fisher s Exact Test. 

Thus, cytotoxicity assays are unlikely to provide an adequate predictor of in vivo irritation in every case. There is no doubt that some type of compounds (such as surfactants) will give (and have given) acceptable correlations with in vivo data. However, for the diversity of compounds common in the pharmaceutical industry, cytotoxicity assays alone are inadequate predictors of ocular irritation, though they may have a place in a battery of tests. For instance, if one is interested in a very quick assessment that will be corroborated later, cytotoxicity assays may be indicated. But each laboratory needs to make its own evaluation of the utility and value of these methods. 

Additionally, the test materials used in the validation process should be as closely related as possible to the characteristics of the unknowns to be tested. It is clear from the literature, for instance, that many cytotoxicity assays give good correlations with the in vivo ocular irritancy data for surfactants, but the correlations fail when compounds from other chemical classes are tested. Since any particular assay may be used differently by individual safety assessment programs, users must evaluate potential methods under conditions likely to be encountered in their own situations. 

Thomson, M.A., Hearn, L.A., Smith, K.T., Teal, J.J. and Dickens, M.S. (1989b). Evaluation of the neutral red cytotoxicity assay as a predictive test for the ocular irritancy potential of cosmetic products. In Alternative Methods in Toxicology, Vol. 7 (Goldberg, A.M., Ed.). Mary Ann Liebert, New York, pp. 297-305. 

After preincubation in RPMI-1640 medium containing 10% fetal calf semm (PCS), the residual cytotoxic activity of the conjugates against colon 26 tumor cells was measured by MTT assay method at 37°C in vitro. 

Shroyer, M., and Bhunia, A. (2003). Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species. /. Microbiol. Methods 55,35-40. 

T. Mosmann, Rapid colorimetry assay for cellular growth and survival application to proliferation and cytotoxic assays, J. Immunol. Methods 65, 55-63 (1983). 

Endpoint assays such as proliferation or cytotoxicity assays are routinely used for functional assessments. For these assessments, primary cells, transformed cells, or cells transfected with the target receptor are exposed to range of concentrations of the test article. Proliferation or cytoxicity is then measured using a variety of methods such as crystal violet vital dye staining, MTT/MTS incorporation, or a luminescence readout like ATP lite. In addition, assays that analyze phosphorylation of specific transcription factors, or release of specific cytokines and chemokines, are also common. Figure 9.5 illustrates the measure of functional consequences of receptor-test article interaction by quantifying cytokine release. Cells from the species under evaluation were cultured in the presence of serial dilutions of the test article or control reagents, and supernatants harvested for determination of cytokine levels by ELISA (i.e.,... 

A number of studies have reported that the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) in vitro cytotoxicity assay is a convenient method for assessing ceU viability. The main features found with this assay are its ease of use, accuracy and rapid indication of toxicity. This method is of interest particularly when exposure to unknown chemical substances requires the rapid detection and evaluation of toxic effects. Direct comparisons of the IC50 value determined in the MTS assay and in vivo cytotoxicity showed a significant, direct correlation. The highest correlation was found when the MTS assay was compared with test systems using human cell hnes. The MTS assay is based on the conversion of a tetrazolium salt into a colored, water-soluble formazan product by mitochondrial activity of viable cells at 37 °C. The amount of formazan produced by dehydrogenase enzymes is directly proportional to the number of living cells in culture and can be measured at 492 nm. 

Borenfreund E Puerner JA (1983) A simple quantitative procedure using monolayer cultures for cytotoxicity assays. Journal of Tissue Culture Methods 9 7-9. 

Twelve 4-trifluoromethylimidazoles (1-12) were synthesized by the method of Kawase et al. [24,25]. Compound 13 was purchased from Aldrich, USA. Cytotoxicity assays and determination of 50% cytotoxic concentration (CC50) against human promyelocytic leukaemia HL-60 and human oral squamous cell carcinoma HSC-3 cell lines were performed as described elsewhere [23]. 

Mosmann T (1983) Rapid colorimetric assay for cellular growth and siu-vival application to proliferation and cytotoxicity assays. J Immunol Methods 65 55-63... 

Kim GGG, Donnenberg VSV, Donnenberg ADA, Gooding WW, Whiteside TLT (2007) A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells comparisons to a 4 h 51Cr-release assay. J Immunol Methods 325 16-66... 

In the following chapter, we introduce HCI assay methods which can be used to identify cytotoxicity, other drug-induced specific toxicologically relevant events, as well as assays for the assessment of cellular morphology and functions. An overview of cellular features associated with potential toxic effects for which specific assays can be applied are shown in Fig. 1. Microscopy images selected from assays are shown in Fig. 2. 

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