Caspase-3 Activity and Cell Necrosis Assay

Caspase-3 activity was measured by using the EnzChek™ Caspase-3 Assay kit purchased from Invitrogen Molecular Probe, using the manufacturer's instructions. Briefly, about 6 × 10⁵ cells were washed with PBS and then lysed in 65 μl of the kit lysis buffer. The caspase-3 substrate Z-DEVD-AMC was added to a final concentration of 200 μM in 50 μl of the kit reaction buffer, which was then mixed to an equivalent volume of cell lysate. Fluoromethylcoumarin fluorescence, resulting from the caspase-3-mediated cleavage of Z-DEVD-AMC, was detected using the POLARstar Omega microplate reader (BMG Labtech) with excitation/emission wavelengths of 350/450 nm with a 45-min kinetics. The remaining cell lysate was used to normalize the fluorescence values to the total protein content. Results were expressed in comparison to nonsteatotic or steatotic untreated cells, as appropriate. Lactate dehydrogenase (LDH) release in the culture medium was used as an indicator of cell necrosis, as previously reported [23 (link), 24 (link)]. To this end, LDH activity was measured in culture medium aliquots using the Lactate Dehydrogenase Assay Kit purchased from Abcam, according to the manufacturer's recommendations. Results were expressed in comparison to nonsteatotic untreated cells.