Cytotoxicity is the quality of being toxic to cells.
Cells exposed to a cytotoxic compound can respond in a number of ways. The
cells may undergo necrosis, in which they lose membrane integrity and die
rapidly as a result of cell lysis; they can stop growing and dividing; or they
can activate a genetic program of controlled cell death, termed apoptosis.
Cytotoxicity testing: measuring viable cells, dead cells,
and detecting mechanism of cell death.
Testing the effects of compounds on the viability of cells
grown in culture is widely used as a predictor of potential toxic effects in
whole animals. Among the several alternative assays available, measuring the
levels of ATP is the most sensitive, reliable, and convenient method for
monitoring active cell metabolism. However, recently developed combinations of
methods have made it possible to collect more information from in vitro
cytotoxicity assays using standard fluorescence and luminescence plate readers.
This chapter describes two assay methods. The first utilizes beetle luciferase
for measuring the levels of ATP as a marker of viable cells. The second more
recently developed multiplex method relies on selective measurement of three
different protease activities as markers for viable, necrotic, and apoptotic
cells. Data analysis from the measurement of three marker protease activities
from the same sample provides a useful tool to help uncover the mechanism of
cell death and can serve as an internal control to help identify assay
artifacts.
