Caspace fitc vad fmk marker

The detection of caspase activity was performed using the CaspACE FITC-VAD-FMK marker (Promega), as described by the manufacturer. The FITC-VAD-FMK marker is an analog of the Z-VAD-FMK caspase inhibitor (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) that enters the cell and irreversibly binds to activated caspases. This test was done as described in the section “Effect of synthetic peptides on yeast growth” with the following differences: after incubation with 25 µM of the smallest and the strongest active peptide for 24 hours, the C. buinensis cells were resuspended, washed once in 500 µL PBS (10 mM NaH₂PO₄, 0.15 M NaCl) with pH 7.4 and resuspended in 50 µL of the staining solution (supplied by the kit) containing 50 µM FITC-VAD-FMK marker. After incubation for 20 minutes at 30°C with constant agitation at 500 rpm, the cells were again washed in 500 µL PBS and resuspended in 20 µL PBS. Negative control (in the absence of the peptide) and positive control cells (incubated with 300 mM acetic acid) had the same treatment as cells treated with the peptide. The cells were analyzed by DIC on the optical microscope equipped with a fluorescence filter for fluorescein detection (excitation wavelength 450–490 nm and emission wavelength 500 nm).