Figure 5.

The specificity of the cleavage of the caspase-sensitive CFP-LEVD-YFP probe by individual caspases was determined using specific caspase inhibitors. A: Hela cells were incubated with various caspase inhibitors as indicated, then were transfected with the caspase-sensitive probe, and analyzed for FRET assay by flow cytometry. A FRET-positive population and a population with diminished FRET were gated in R3 and R4, respectively. The mean fluorescence intensities (MFI) and percentage of each population compared to all gated CFP/YFP double-positive cells are shown in each panel. To quantitate caspase activity (CA_f) by flow cytometry, caspase activity was calculated by the formula: CA_f = (FRET_bright − FRET_dim)/FRET_bright × 100 × FRET_dim, where FRET_bright and FRET_dim are the mean fluorescence intensities of FRET in the FRET_bright cells and the cells with diminished FRET, respectively; and where %FRET_dim is the percentage of cells with diminished FRET among all transfected cells. B: The FRET-based caspase activity in living cells when incubated with specific caspase inhibitors.