Caspase glo 8 and 3 7 assay systems

Caspase 8 and caspase 3/7 activity was measured using luminescent assay kits (Caspase-Glo 8 and 3/7 Assay Systems, Promega, Madison, WI, USA, Cat Nos. G8200 and G8090, respectively). Twenty-four hours prior to treatment, 661W cells were seeded in white-walled 96-well plates at 2500 cells/well. Cells were treated for 2 h with compound 2 or DMSO prior to the addition of 500 ng/mL FasL (Recombinant Mouse Fas Ligand/TNFSF6 Protein, R&D Systems Inc., Minneapolis, MN, USA, Cat No. 6128-SA-025) and 250 ng/mL HA (Hemagglutinin/HA Peptide Antibody, R&D Systems Inc., Minneapolis, MN, USA, Cat No. MAB060) [9 (link)]. As previously published, caspase activity was measured 8 h after treatment by incubating cells with substrate for 1 h as per the manufacturer’s instructions. A plate reader luminometer (BMG Labtech, Inc., Cary, NC, USA) assessed luminescence [9 (link),38 (link)].

Luminescent assay kits (Caspase-Glo 8 and 3/7 Assay Systems, Promega, Madison, WI, Cat Nos. G8200 and G8090, respectively) were utilized to measure caspase 8 and caspase 3/7 activity according to the manufacturer’s instructions. The 661 W cells were seeded in white-walled 96-well plates at 2,500 cells/well for 24 hours prior to treatment. Cells were pre-treated with ML-265 or DMSO for 2 hours prior to treatment with 500 ng/ml FasL (Recombinant Mouse Fas Ligand/TNFSF6 Protein, Cat No. 6128-SA-025, R&D Systems Inc., Minneapolis, MN, USA) and 250 ng/ml HA (Hemagglutinin/HA Peptide Antibody, Cat No. MAB060, R&D Systems Inc., Minneapolis, MN, USA). Similar to that previously described, caspase activity was measured at 8 hours status post treatment by incubating the cells with substrate for 1 hour according to the manufacturer’s instructions. Luminescence was measured in a plate reader luminometer (BMG Labtech, Inc, Cary, NC)^{36 (link)}.

Caspase‐8 and caspase‐3/7 activity assays were performed using Caspase‐Glo 8 and 3/7 Assay Systems (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, 1.5 × 10⁴Smg7^−/− or parental MF cells per well were seeded 1 day before in a 96‐well plate. On the second day, cells were exposed to 20 ng·mL⁻¹ TNFα (Thermo Fisher Scientific) for 8 h or left untreated. The medium was removed, and cells were washed twice with PBS. Fifty microlitre reagent and 50 µL PBS were added and incubated at room temperature with continuous shaking. After 20 min, luminescence was recorded on an Envision 2104 Multilabel plate reader (PerkinElmer). Empty wells served as blanks and were subtracted from all values.