Flow cytometric assay for evaluation of the effects of cell density on cytotoxicity and induction of apoptosis

Abstract

We used a flow cytometric assay, which allows us to perform precise measurements within a wide range of cell concentrations to study the effect of the density of cultured cells on their sensitivity to cytotoxic compounds. To measure cytotoxic action, cells are plated in a 96-well plate at a density ranging from 700 to 100,000 cells/ml and are allowed to grow for 72 h in the presence of various concentrations of a cytotoxic agent. To quantitate the number of surviving cells, each sample is analyzed in a flow cytometer with equal acquisition time. Viable cells are identified by light scattering characteristics identical to those for untreated cells. To estimate the amount of viable, apoptotic, or necrotic (late apoptotic) cells, the samples are stained with Annexin V and propidium iodide. Using this method, we found that the cytotoxicity of ascorbic acid for malignant lymphoid CEM-C7 cells can be increased significantly when cell density decreases, reaching a value that is typically lower than the normal physiological concentration of ascorbic acid in blood. The flow cytometric analysis described in this study can be useful in comparing the effects of cell density on the cytotoxic action of various compounds.