Apoptosis Studies

Cells were pretreated with 10 µM etoposide or DMSO in 5% RPMI for 24 h. Subsequently, cell apoptosis was measured with a microplate reader–based TiterTACS in situ apoptosis detection kit (R&D systems; 4822-96-K) as described by the manufacturer. Percentage rise in apoptosis was derived from the difference in absorbance between the etoposide-treated and DMSO-treated wells. Minimum of duplicates were performed for each experiment.

Cells were pretreated with 10 µM etoposide or DMSO in 5% RPMI for 6 h before incubation in fresh medium for up to 24 h. Subsequently, the cells were harvested and stained with Annexin V and propidium iodide according to manufacturer's protocol (BD Biosciences; 556547). Cells in early apoptosis are Annexin V positive and PI negative, whereas cells in late apoptosis or necrosis are both Annexin V and PI positive. For our study, we only considered apoptotic cells with Annexin V positive and PI negative. Cells with double staining were excluded, given that some of these cells might have died via necrosis. A minimum of triplicates was performed for each experiment.