Assessment of Cell Viability

The cells were stained with the DNA-binding dyes propidium iodide (PI), 5 µg/mL, and Hoechst 33342 (HO), 5 µg/mL, (Sigma). The number of viable, apoptotic, and necrotic cells was determined by 2 independent observers, one of them unaware of sample identity. The agreement among observers counting was > 90%. In each experimental condition, at least 600 cells were counted. Results are presented as percentage of apoptosis, calculated as number of apoptotic cells/total number of cells ×100, or as percentage of total cell death, calculated as number of apoptotic plus necrotic cells/total number of cells ×100. This method has been validated for use in β cells by systematic comparison with different techniques including electron microscopy, cleavage of the effector caspase 3, and DNA laddering [27, 28]. In selected experiments, apoptosis was confirmed by activation (cleavage) of caspase 3 by Western blot.