Quantifying Caspase-3 Activity in BMDMs

To assess caspase‐3 activity, 1 × 10⁵ BMDMs were plated per well in 96‐well flat‐bottom tissue culture‐treated plates (BD Falcon), primed with LPS (50 ng/ml) for 3 h and then treated, as indicated with Q‐VD‐OPh (40 μM), ABT‐737 (500 nM) and S63845 (10 μM). All stimulations were staggered in a reverse time course fashion to ensure identical LPS priming and QVD incubation periods across treatments. At experimental endpoint, supernatants were discarded after cell centrifugation and BMDMs lysed in 70 μl DISC lysis buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% TritonX‐100, 10% Glycerol, H₂O) for 1 h at room temperature with rotation. 10 μl of cell lysate was used for protein quantification (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific) according to manufacturer's instruction, and 50 μl was transferred to an opaque‐walled, clear bottom 96‐well flat bottom plate. DEVDase substrate (Ac‐DEVD‐AMC, BD Pharmingen™ Cat) was prepared to working concentration (20 μM) in assay buffer (20 mM HEPES pH 7.5, 10% Glycerol, 2 mM Dithiothreitol (DTT)). 200 μl of prepared DEVDase substrate reagent was added to each well containing lysates and allowed to incubate overnight with rotation in the absence of light. After incubation, caspase AMC fluorescence was detected using a CLARIOstar Plus (BMG Labtech). Protein concentrations obtained from the BCA were used to normalise the AMC fluorescence intensity across samples.