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Caspase activation assay

SM Stephanie McKenna
LG Lucía García-Gutiérrez
DM David Matallanas
DF Dirk Fey
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Caspase activation was measured in an Accuri's C6 Flow Cytometer System using the CaspACE-FITC-VAD-FMK kit (Promega). Alive and dead cells were collected, centrifuged for 5 min at 300×g and incubated with FITC-VAV-FMK in serum free media (1:2000) for 30 min at 37 °C protected from light. Cells were washed once with PBS and incubated with PI for 5 min at room temperature prior to acquisition. FITC and PI emissions were measured using the 533/30 and 670LP detectors respectively following excitation by 488 nm laser. Statistical analysis was carried out to determine the mean caspase activation and standard deviation within each group (siNT, siBAX and siSMAC), control and treated. Fold change in caspase activation was calculated for each treated group compared to the group control.

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