Elp il1b 1

J774A.1 or PAMs cells were stimulated by H. parasuis or OMVs as indicated. After 24 or 48 h, supernatants from cell culture were harvested and assayed with the production of murine IL-1β (R&D Systems, Cat# MLB00C) and swine IL-1β (RayBio, Cat# ELP-IL1b-1) by enzyme-linked immunosorbent assay, in accordance with the manufacturer's instructions. The absorbance was quantified at 450 nm by a microplate spectrophotometer (Synergy H1, BioTek, USA).

PAMs were seeded in 24-well cell culture plates for 12 h, then infected with ASFV-WT or Del2R for 1 h and rinsed twice with PBS. The infected cells were cultured in the fresh RPMI 1640 medium for 12 and 20 h. The concentrations of IL-1β (Ray Biotech, ELP-IL1b-1), TNF-α (Ray Biotech, ELP-TNFa-1), and IFN-α (Ray Biotech, ELP-IFNa-1) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. The cells were collected for RT-qPCR analysis as described above, and the mock-infected cells were included as a negative control.

PAMs were infected with Del4L or ASFV-WT at an MOI of 5. The expression of IL-1β (Ray Biotech, catalog no. ELP-IL1b-1), TNF-α (Ray Biotech, catalog no. ELP-TNFa-1), and IFN-α (Ray Biotech, catalog no. ELP-IFNa-1) in the cell culture supernatants was measured by ELISA kits according to the manufacturer’s protocols at 12 and 20 hpi. The concentrations of these cytokines were determined based on standard curves.