Il 1β enzyme linked immunosorbent assay elisa kit

LPS (E. coil O111:B4) used for cell culture studies was purchased from Calbiochem (San Diego, CA, USA; cat# 437627), and for animal studies, it was purchased from Sigma-Aldrich (St. Louis, MO, USA; cat# L3012). IL-10, tumor necrosis factor alpha (TNFα), and interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kits and salmeterol (a long-acting selective β2-adrenergic receptor agonist) were obtained from R&D Systems (Minneapolis, MN, USA). Recombinant mouse IL-10, Ultra-LEAF™ Purified anti-mouse IL-10 antibody, and isotype IgG were from Biolegend (San Diego, CA, USA). The anti-mouse IL-10 antibody from Biolegend was used as the detecting/capture antibody for ELISA/ enzyme-linked immunospot (ELISPOT) assay and for neutralization of mouse IL-10 bioactivity in vivo and in vitro (https://www.biolegend.com/en-us/products/ultra-leaf-purified-anti-mouse-il-10-antibody-17764, accessed on 6 August 2021). The specificity and utility of this antibody were further validated in our laboratory. We found that enhanced IL-10 protein level was detected in the supernatant of LPS-stimulated mouse primary cell cultures by the R&D IL-10 ELISA kit, but no significant difference between vehicle and treatment with LPS plus anti-mouse IL-10 antibody was observed.

BOT-4-one was provided by Sang-Kyu Ye and Byung-Hak Kim (Seoul National University of College of Medicine, South Korea)^{17 (link)}. Penicillin-streptomycin, fetal bovine serum (FBS), opti-MEM, and RPMI 1640 were purchased from Gibco (Grand Island, NY, USA). IL-1β antibody (AF401)⁸ and IL-1β enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against ASC (AL177)⁸ , NLRP3 (Cryo-2)⁸ , and caspase-1 (clone Casper-1)⁸ were purchased from Adipogen (San Diego, CA, USA). β-actin (sc-1616)^{43 (link)} and ubiquitin (sc-8017)^{37 (link)} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monosodium urate (MSU) crystal, Bay11-7082, silica crystal, nigericin, flagellin from S. typhimurium, poly(dAdT), Pam3CSK4, and zVAD-FMK were purchased from InvivoGen (San Diego, CA, USA). Mouse TNF-α ELISA Ready-SET-Go was purchased from eBioscience (San Diego, CA, USA). A western blot chemiluminescence reagent kit was purchased from Pierce Chemical (Rockford, IL, USA). Polyvinylidene fluoride (PVDF) and nitrocellulose membrane were purchased from Millipore Corporation (Bedford, MA, USA). LPS (Escherichia. coli 026:B6 and 011:B4), 3,4-methylenedioxy-β-nitrostyrene, and ATP were purchased from Sigma-Aldrich (St.Louis, MO, USA). All other chemicals used were of the highest quality among those that are commercially available.