Quantifying Caspase 3 Activation via Flow Cytometry

Percentage of cells showing Caspase 3 activation was determined by FITC-VAD-FMK staining, followed by flow cytometry quantification as previously described [46 (link)]. Briefly, both floating death cells and attached cells were collected, centrifuged 300 × g and resuspended in 5 µM FITC-VAD-FMK (CaspACE™ FITC-VAD-FMK, Promega) containing serum-free media and incubated at 37 °C for 30 minutes protected from light. Cells were washed once with PBS, resuspended in PBS containing 0.5 µg/mL of PI (Biolegend, CA USA) and incubated at room temperature for 10 min protected from light. Samples were acquired and analysed with a BD Accuri^TM C6 Flow Cytometer (MA, USA). FITC-VAD-FMK fluorescence was exited using a 488 nm laser and detected with the FL1 525/25 nm filter. PI fluorescence was excited using a 488 nm laser and detected with the FL2 585/40 nm filter.