Caspase 8 assay kit

Caspase-3, caspase-8, and caspase-9 activity was measured using a Caspase-3 Assay Kit, Caspase-8 Assay Kit, and Caspase-9 Assay Kit (Beyotime, Shanghai, China), respectively. A 6-well cell culture plate was inoculated with 1 × 10⁶ RAW264.7 cells per well and incubated for 16 h. WT, ΔsseK3 mutant, and sseK3-complemented strains were incubated with the RAW264.7 cells at a MOI of 100:1, with three replicate wells per strain. The plates were centrifuged at 1000 rpm and gentamicin-containing medium (100 μg/mL) was added and incubated at 37 °C with 5% CO₂. The supernatants were then aspirated, and the cells were washed three times with PBS. Subsequently, the cells of the infected and mock groups were digested by trypsinization without EDTA and washed three times with ice-cold lysis buffer, and treated with 100 μL lysis buffer on ice. After incubation for 15 min, the concentration of protein was detected using the Bradford protein assay kit (Beyotime, Shanghai, China). Subsequently, the cell lysates were incubated with Ac-DEVD-pNA for 4 h at 37 °C, the absorbance was read at 405 nm in a microplate spectrophotometer (Infinite 200 PRO NanoQuant, Tecan, Switzerland).

Cells (1 × 10⁴ cells/well) were cultured in 96-well plates overnight and infected in triplicate with, or without, viruses at a MOI of 5 for 24 hours. The levels of caspase 3/7/8 activities in individual wells were measured by luminescent assay using the Caspase-Glo 3/7 kit (#G8091, Promega, Madison, WI, USA), caspase 8 assay kit (C1151, Beyotime, Shanghai, China) in a Synergy HTX Multi-Mode reader (BioTek, VT, USA).