Caspase 1 ice colorimetric assay kit

For caspase-1 activity assay, caspase-1 activity was measured using caspase-1/ICE colorimetric assay kit (Biovision Inc, #K111) according to manufacturer’s protocol.

Caspase 1 activity was determined using a caspase 1/ICE colorimetric assay kit (BioVision, USA). Once the treatment was terminated, the cell lysates were collected using chilled cell lysis buffer, and the supernatant was collected after centrifugation. The protein concentration was determined with a BCA protein assay kit (Beyotime), and 100–200 μg of protein from each sample was incubated with reaction buffer and YVAD-ρNA substrate at 37 °C for 2 h. The samples were read at 405 nm using the Multiskan FC (Thermo Scientific).

This was carried out on pelleted PBMCs after incubation with medium (control) and PRRSV strains, respectively, using caspase-1/ICE Colorimetric Assay Kit (BioVision, catalog number K111-200) as specified by the manufacturer, with minor changes and under strict cold chain conditions. Briefly, 50 μl/well of the kit's cell lysis buffer was added to the cell pellet, and plates were incubated for 10 min over crushed ice. Plates were again centrifuged at 2,200 g, 10 min, 4°C. Each supernatant was transferred to a well of an ELISA plate (NUNC Maxisorp flat-bottom, catalog number 44-2404-21, Thermo Fisher Scientific) and reacted with 50 μl/well of the kit reaction buffer. This was also added to the positive control (1 μl caspase-1 standard + 49 μl of reaction buffer) and to the negative control (50 μl/well of cell lysis buffer). Then, 5 μl/well was added of chromogen YVAD-pNA. Plates were incubated at 37°C, 5% CO₂ per 1.5 h and read spectrophotometrically at 405 nm. Results were expressed in terms of DeltaOD at 405 nm, (i.e., OD_405nm sample – OD_405nm negative control).