Caspase-3 activity assays

Caspase-3 activity was measured by using the caspase-3 colorimetric assay kit following the procedure provided by the manufacturer. Briefly, treated and untreated cells were collected after 48-h treatment and re-suspended in ready to use chilled lysis buffer for 10 min. Next, high-speed centrifugation was performed and the supernatants were collected and used for caspase-3 activation assay. Briefly, reaction buffer, DTT and DEVD-p-NA substrate were added to samples and plates were incubated at 37 °C for 2 h. The principle was that caspase-3 derived from cellular lysate recognizes the sequence Asp-Glu-Val-Asp (DEVD). The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the labeled substrate (DEVD-p-NA). The p-NA light emission can be quantified using a microtiter plate reader at 405 nm. Comparison of the absorbance of p-NA from an apoptotic sample with an untreated control sample allows determination of the fold increase in caspase-3 activity. Procedure measures only the functionally relevant cleaved caspase-3.