Chapter 3 Cell Cycle Phase-Specific Analysis of Cell Viability Using Hoechst 33342 and Propidium Iodide after Ethanol Preservation

Abstract

This chapter discusses several techniques that have been developed to quantitate cell viability utilizing the differential staining of fluorescent dyes. Propidium iodide (PI), ethidium bromide (EB), fluorescein diacetate (FDA), acridine orange (AO), erythrosin B, and diamidinophenylindole (DAPI) have been used to quantitate cell viability, and some of these have been used in flow cytometric (FCM) assays. The permeability of dying cells to PI or EB is analogous to the staining by trypan blue; PI is excluded from viable cells. FDA has also been applied to FCM analysis. A nonpolar nonfluorescent fatty acid ester, FDA accumulates intracellularly in viable cells after it is hydrolyzed by esterases, yielding free fluorescein. The free fluorescein is polar and is trapped intracellularly. Dead and dying cells remain nonfluorescent or weakly fluorescent. EB suppresses the DNA-specific green AO fluorescence and permits earlier identification of damaged cells. Using this technique, two steps were observed in the kinetics of cell death following treatment with various chemotherapeutic agents and hyperthermia.