5.6. Caspase-3 Activity Assay

Apoptosis was assessed using the Caspase-3 kit according to the manufacturer’s recommendations (Roche Molecular Biochemicals, Pleasanton, CA, USA). Briefly, HK-2 cells treated with venom or SMase D (10 µg/10⁶ cells), for 2 h at 37 °C, were lysed with the lysis buffer and centrifuged for 1 min, 20 °C at 14,000 rpm. The supernatant was removed and stored at −20 °C until use. 96-well plates were sensitized with anti-caspase-3 monoclonal antibody and incubated at 4 °C overnight. Blocking solution was added to all wells and the plates were incubated for 30 min at room temperature. The plates were washed with wash solution and 100 μL of each experimental sample were added and the plates were incubated for 1 h at 37 °C. The plates were washed, substrate solution (Ac-DEVD-AFC) was added to the wells and the plates were incubated at 37 °C. Activation of Caspase-3 was determined after 3 h using a VICTOR3™ spectrofluorimeter (Perkin-Elmer, Waltham, MA, USA), using excitation and emission wavelengths of 405 nm 535 nm, respectively. The Caspase-3 activity was expressed as the amount of Caspase-3-induced release of the fluorescent AFC (7-amido-4-trifluoromethyl coumarin) per minute.