Quantifying Apoptosis: Nuclear Morphology and Caspase Activity

Apoptosis was analyzed via assessing percent apoptotic nuclei and caspase 3/7 activity, which represent structural and biochemical markers of apoptosis, respectively. Percent apoptotic nuclei was quantified by characteristic nuclear morphology and visualized with the treatment of DNA binding fluorescent dye, DAPI (4’, 6-diamidine-2-phenylindole dihydrochloride) [14 (link)]. Briefly, cells were stained with DAPI (5 µg/ml) for 10–15 min at 37 °C. Apoptotic cells, characterized by condensed and fragmented nuclei were counted and presented as percent of total nuclei. Experiments were performed in triplicates and at least 200 cells were counted per well. Caspase 3 and 7 activity was analyzed using rhodamine 110 bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide) (Z-DEVD-R110) caspase substrate according to manufacturer’s instructions (Promega, Madison, WI #G7791). Briefly, cells with active caspase 3/7 enzyme cleaves the substrate Z-DEVD releasing rhodamine which was quantified spectrofluorometrically using Biotek Synergy plate reader [14 (link)]. The experiment was performed in quadruplicate and were reported as fold change compared to vehicle-treated cells.