Assay for Cytotoxicity

The procedures used for AG selection in situ have been described in full. The term in situ refers to procedures in which, for each experimental point, a 

HPRT diploid human fibroblasts are very efficient at transferring the phosphorylated nucleoside of AG by cell-to-cell contact into HPRT cells (metabolic cooperation),which renders the HPRT cells sensitive to killing by AG despite their genotype. Therefore, it is necessary to make use of large numbers of dishes to obtain sufficient surface area so that the cells will not be too close to each other at the end of the expression period. Thus, for the in situ technique, 500-1000 60-mm-diameter dishes were commonly used for a mutagenesis experiment involving a control and two or three experimental points, and each of these had to be exposed to UV radiation or carcinogen. Furthermore, since AG is unstable in the presence of calf serum, 

Just as with the in situ technique, reconstruction experiments with HPRT Lesch-Nyhan (LN) cells accompanied each replating mutagenesis experiment to determine the efficiency of recovery of AG- or TG-resistant cells in the presence of nonresistant cells. That is, for each individual determination, a known number of LN cells (CRL 1112, American Type Culture Collection) were seeded into flasks or dishes containing either no other cells or cells seeded at the same density as in the experimental dishes or bulk culture vessels. The cloning efficiency of the LN cells in the presence of the experimental cells, divided by their cloning efficiency when seeded into empty dishes, determined the efficiency of recovery of mutant colonies. Our studies with the in situ technique, in which loosely formed miniature colonies are present at the time selection with AG begins, showed that if the total number of cells present in 60-mm-diameter dishes reached 30,000, recovery decreased to approximately 65%. With the replating technique, in which individual cells are plated directly into selective medium at the end of the expression period, a density of 500-750 attached cells/cm for TG and 1000-1500/cm in AG resulted in approximately 85% recovery (data not shown). Therefore, cells were plated at or below these respective lower densities. 

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Sina, J.E, Bean, C.L., Bland, J.A., MacDonald, J.J., Noble, C., Robertson, R.T. and Bradley, M.O. (1986). An in vitro assay for cytotoxicity to proximal tubule suspensions from rabbit kidney. In Vitro Toxicol. 1(1) 12-22. 

B Page, C Page, M Noel. A new fluorometric assay for cytotoxicity measurements in vitro. Int J Oncol 3 473-476, 1993. 

Suffness, S.M. and Pezzuto, J. (1991). Assays for cytotoxicity and antitumor activity , In Methods in Plant Biochemistry, Hostettmann, K. (Ed.), London Academic Press, Vol 6 pp. 71-133. 

Before a compound can be tested for antiviral activity, it must first be assayed for cytotoxic effects on the uninfected cells at the same concentration. 

Berberine chloride, as well as a series of twenty other pharmacologically active agents, was evaluated in a new microplate assay for cytotoxicity using the brine shrimp Artemia salina, and shown to furnish comparable results to those previously published in the test-tube method. The assay reliably detected all of the compounds toxic to KB cells in a series of twenty-one pharmacologically active agents, except for two which required metabolic activation in humans [241],... 

Growth inhibition assessed by sulforhodamine B colorimetric assay for cytotoxic effects. All values are mean standard deviation, N/A = not active. 

Rodgers KE, Leung N, Imamura T, et al. 1986. Rapid in vitro screening assay for immunotoxic effects of organophosphorus and carbamate insecticides on the generation of cytotoxic T lymphocyte responses. Pestic Biochem Physiol 26 292-301. 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

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. 

Borenfruend, E. and Puemer, J.A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays. J. Tissue Culture Meth. 9 7-9. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

Skehan, P. et al., New colorimetric cytotoxicity assay for anticancer-drug screening, /. Natl. Cancer Inst., 82, 1107-1112, 1990. 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

Figure 4 Cytotoxicity of HER2-directed liposomal APE in HER2-overexpressing BT474 human breast carcinoma cells. Cells were plated at a density of 5000 ceUs/well and incubated for four hours with varying concentrations of unencapsulated ( ), liposomal (O), or antiHER2 (E5)-immunoliposomal (A) Cells were assayed for viability using a standard tetrazolium-based assay three days later. Abbreviation. APE, 6-(3-aminopropyl)ellipticine.

The first functional cell-based assay for the determination of pan-histone deacetylase activity was achieved by detecting hyperacetylated histones by Western blotting. The histone acetylation level in cells offers a measure of candidate HDACI activity and has been linked to antiproliferative and cytotoxic effects. Beckers et al. used a cellular histone hyperacetylation assay for quantification of the cellular efficacy of HDACIs using the Cellomics Array Scan II platform in 96 wells [14]. The program... 

Cytotoxicity Evaluated for confounding interpretation of in vitro efficacy assays, for predicting potential for human toxicity especially in liver but also if warranted by other safety assessments in bone marrow, kidney, neurons, immu-nocytes and so on. Also used for developing understanding of biochemical mechanisms of toxicity. HCA has been repeatedly demonstrated to be an effective tool in predictive toxicology. May also be used for certain translational safety biomarkers of toxicity [37]... 

Effective Cell-Based Assays for Marked and Acute Cytotoxicity... 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

Xu, J.J., Diaz, D. and O Brien, P.J. (2004) Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chemico-Biological Interactions, 150, 115—128. 

Repetto, G., del Peso, A. and Zurita, J.L. (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols, 3, 1125-1131. 

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