Caspase 3 7 assay kit

Cells for flow cytometry were counted and 5×10⁵–1×10⁶ cells were placed in individual 12 × 75 mm round-bottom tubes suspended in flow cytometry staining buffer (Invitrogen, 00–4222-26). For apoptosis, a Caspase-3/7 assay kit was used (Invitrogen, C10427). Each tube was brought to a volume of 1 ml staining buffer and 1 μl of CellEvent Caspase-3/7 reagent was added. Cells were incubated at 37 °C in the dark for 25 minutes and then 1 μl of SYTOX AADvanced dead stain solution was added. Cells were incubated at 37 °C for 5 minutes and then analyzed. For cell cycle analysis, cells were fixed with 0.5 ml of 100% cold ethanol for 20 minutes at 4 °C under gentle rotation. Cells were pelleted and the ethanol was decanted. Cells were resuspended in 1 ml of staining buffer and 4 drops (164 μl) of Propidium Iodide Ready Flow (Invitrogen, R37169) was added to the cells and incubated at 25 °C for 20 minutes. Cells were analyzed on a ZE5 Cell Analyzer (Bio-Rad) and data processed using FCS Express 6 Flow Cytometry software (De Novo Software, Pasadena, CA). The percentage of the cell population in G1, or S/G2 phase of the cell cycle was calculated by fitting Propidium Iodide excitation counts using FCS Express: Multicycle (De Novo Software).