Caspase glo 8 assay kit

Cells (1 × 10⁴ cells/well, 96-well plates) were infected with viruses at MOI 10 for 6 h or 24 h (n = 3). Caspase activity was measured using ApoTox-Glo™ Triplex (caspase-3/7) and Caspase-Glo-8 assay kits (Promega, Madison, WI). Luminescence signal was measured using a Synergy HTX Multi-Mode reader and the data was normalized by background subtraction and in relation to uninfected cells (n = 3, biological replicates). Mitochondrial membrane potential (ΔΨm) assay: Cells were infected with viruses at an MOI 10 for 48 h. The MitoTracker™ Deep Red FM kit (Thermo Fisher Scientific) was used to measure mitochondrial membrane potential (n = 3, biological replicates).

Caspases-3, -7, and -8 activities were determined by using a Caspase-Glo 3/7 or a Caspase-Glo 8 assay kit (Promega) according to the manufacturer's instructions. In brief, the cells were seeded at a density of 1.0 × 10⁴ cells/well in a 96-well plate. After drug treatment, an equal volume of Caspase-Glo 3/7 or Caspase-Glo 8 reagent was added to the cell culture medium, which had been equilibrated to room temperature for 1 hour. Cells were shaken for 5 min and incubated at room temperature for 30 min. Luminescent recording was performed with a Synergy H1 microplate reader (BioTek).

The quantification of caspase enzymatic activity is regarded as an important readout for apoptosis. We used the Caspase-Glo 3/7 Assay Kit, the Caspase-Glo 8 Assay Kit, and the Caspase-Glo 9 Assay Kit (Promega Co., USA) to examine the activity of caspase-3/7, caspase-8, and caspase-9, respectively, according to the manufacturer’s protocol. Briefly, A2780 and SKOV3 cells were plated at a density of 4 × 10⁵ and 3.5 × 10⁵ cells per well in 6-well plates, respectively. Following the indicated treatments (1.25–5 μM JS-K; vehicle, 2.5 μM JS-K, 200 μM NAC, co-treatment with 2.5 μM JS-K and 200 μM NAC) and incubation period (48 h), approximately 6 × 10⁴ cells per sample were transferred into 1.5-mL tubes. Next, 100 μL of Gaspase-Glo reagent (relative Caspase substrate) was added to each sample, and the cells were incubated for 1 h at RT protected from light. Sample luminescence values were measured using the Sirius L Tube Luminometer (Berthold Detection Systems, Germany). Relative caspase activities were normalized to the raw luminescence units (RLUs) of the untreated control.

Caspase-8 activity was measured using a Caspase-Glo 8 assay kit (Promega). OVCAR-3 or HeLa cells were treated with R27T (100 ng/mL), cFLIP siRNA (10 nM), and/or TBB (10 μM) alone or in combination, and then lysed on ice in phosphate buffered saline containing 1% NP40 and 0.1% SDS. Ten micrograms of cell lysate in a 50 μL total volume was resuspended in 50 μL of Caspase-Glo reagent and incubated for 1 h at room temperature. The luminescence produced upon cleavage of the luminogenic substrate (which contained the caspase-8 cleavage site, LETD) was measured for each sample using a microplate reader (TECAN).

Lumbar spinal cord tissue of naïve or day 12 of EAE WT and cIAP2^−/− mice were flash-frozen in liquid nitrogen, mechanically powdered, and resuspended in 450 μl of cold extraction buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA pH 7.5). Samples were sonicated, centrifuged, and the supernatants (100 μg protein/assay) were used for the assessment of caspase-8 activity using Caspase-Glo^® 8 Assay kit (Promega). Results were expressed as relative luminescent units per 100 μg protein.