Caspase 3 fluorimetric assay kit

The caspase-3 enzymatic activity was measured by using a caspase-3 fluorimetric assay kit (BioVision, Milpitas, CA, USA), according to the manufacturer’s protocol. Briefly, cells were seeded into 75 cm² plates (3 × 10⁶ cells/plate) and after 24 h were treated for 7 h with different concentrations of celecoxib or 0.5% (v/v) DMSO as control vehicle. At the end of drug incubation, cells were collected, washed with phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) constituted by 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4, supplemented with 137 mM NaCl and 2.7 mM KCl, and finally lysed at 4 °C in cell lysis buffer. Cell lysates were incubated with 50 µM DEVD-AFC substrates at 37 °C for 2 h, to detect caspase-3 activity, using a Cary Eclipse spectrofluorometer. Excitation and emission wavelengths were set at 400 nm and 505 nm, respectively; both excitation and emission slits were set at 10 nm.

Full cell lysates generated by lysis in RIPA buffer were mixed with caspase reaction buffer (100 mM Hepes pH 7.4, 10% Glycerol, 0.1% CHAPS, 10 mM DTT and 100 μM Ac-DEVD-AFC (from the Caspase-3 Fluorimetric Assay Kit (Biovision K105-400)). Caspase activity (activity/min/mg of protein) was calculated from a 1 h kinetic cycle reading on a spectrofluorometer (380 nm/460 nm or 405 nm/510 nm, Infinite F500, Tecan).