Assays and Reagents for Measuring Cytotoxicity, Proliferation and Viability

Assays to measure proliferation, viability and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity or DNA synthesis. Cell counting using viability dyes such as trypan blue or calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells. 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) is a popular choice for measuring the number of cellular divisions a population has undergone. Upon entering the cell, CFSE is cleaved by intracellular esterases to form the fluorescent compound and the succinimidyl ester group covalently reacts with primary amines on intracellular proteins. Upon division, the fluorescence intensity of each daughter cell is halved which allows for the simple detection of the number of cell divisions by flow cytometry. Assays that measure metabolic activity are suitable for analyzing proliferation, viability, and cytotoxicity. The reduction of tetrazolium salts such as MTT and XTT to colored formazan compounds or the bioreduction of resazurin only occurs in metabolically active cells. Actively proliferating cells increase their metabolic activity while cells exposed to toxins will have decreased activity.