Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
Count cells and pellet 1-5 x 106 cells or use 50-200 μg cell lysates if protein concentration has been measured.
Resuspend cells in 50 μl of chilled Cell Lysis Buffer.
Incubate cells on ice for 10 minutes.
Add 50 μl of 2x Reaction Buffer and 1 μl DTT to each sample.
Add 5 μl of the 1 mM AFC conjugated substrates (50 μM final conc.) into each tube individually and incubate at 37°C for 1-2 hours.
Read samples in a fluorometer equipped with a 400 nm excitation filter and 505 nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96-well plate.
Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control.