Il 1f2 quantikine elisa kits

Recombinant human TNF-α protein and human IL-1β/IL-1F2 Quantikine ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). ATP, apyrase, anti-β-actin (#A2066) antibody, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-NLRP3 (#ab214185), anti-NLRC4 (#ab99860), and anti-cleaved caspase-1 (#ab179515) antibodies and human VEGF-A ELISA kits were purchased from Abcam (Cambridge, United Kingdom), and anti-ASC (#13833) antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). TOPscript One-step RT PCR Drymix was purchased from Enzynomics (Daejeon, Korea). Negative control siRNA (control siRNA) and NLRP3, NLRC4, ASC, caspase-1, and P2Y₂R siRNA were obtained from Bioneer (Daejeon, Korea), and Lipofectamine 3000 reagent, TRIzol reagent, and anti-P2Y₂R (#PA1-46150) antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hybond-P⁺ polyvinylidene difluoride membrane was obtained from GE Healthcare (Chicago, IL, USA), and Clarity Western enhanced chemiluminescence (ECL) substrate was purchased from Bio-Rad (Hercules, CA, USA). BD Matrigel basement membrane matrix (Matrigel) was obtained from BD Bioscience (Franklin Lakes, NJ, USA), and cell culture inserts for 24-well plates (8.0 µm, Translucent PET membrane) were purchased from Corning (Corning, NY, USA).

Concentrations of human IL-1β in culture supernatant were measured using human IL-1β/IL-1F2 Quantikine ELISA kits according to the manufacturer’s instructions (DLB50; R&D systems, Minneapolis, MN, USA). Briefly, 8.0 × 10⁵ 293T cells in 2 mL DMEM complete medium were seeded in each well of a six-well plate. The supernatants of cells transfected with pEF1α-DEST expressing C-terminal c-Myc-tagged SARS-CoV-2 ORFs were harvested, and 30 μL of each supernatant were diluted 1:10 with 270 μL DMEM complete medium. A 200 μL aliquot of each diluted supernatant was used for ELISA. To measure IL-1β concentrations in the supernatants of THP-1 cells, cells were suspended at 2 × 10⁷ cells/mL in a 110 μL R buffer, and 100 μL of cells electroporated with DNA were added to each well of a six-well plate, along with 2 mL RPMI complete medium (without antibiotics). A 200μL aliquot of each supernatant was used for ELISA. Concentrations of mature human IL-1β were calculated using a standard curve.