Monosodium urate msu crystal

Monosodium urate (MSU) crystals were obtained from InvivoGen (Carlsbad, CA, USA). Phorbol 12-myristate 13-acetate (PMA), colchicine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO; ≥99.5%) were bought from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for IL-1β (12242) and NLRP3 (15101) were purchased from Cell Signaling Technology (USA). Antibodies against caspase-1 (sc-56036), phospho-IκB-α (sc-52943) and NF-κB p65 (sc-8008) were from Santa Cruz Biotechnology (Dallas, TX, USA) and β-actin antibody was from Abcam (Cambridge, UK). Peroxidase-conjugated AffiniPure goat anti-mouse IgG (115-035-003), and anti-rabbit IgG HRP-linked antibody (7074) as secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and Cell Signaling Technology, respectively.

Murine macrophages (3-6x10⁴ cells/ml) were seeded in 500 µl of macrophage media in 24-well tissue culture plates containing acid washed glass coverslips, followed by incubation at 37°C with 5% CO₂ for 30-60 minutes. After optimization of stimulant doses, cells received no treatment or were treated with 5x10⁶ heat-killed Candida albicans (InvivoGen), 20 μM ionomycin (MilliporeSigma), 30 μg/mL LPS from E. coli (InvivoGen), 1.2 mg/mL monosodium urate (MSU) crystals (InvivoGen), 10 μg/mL platelet activating factor (PAF, MilliporeSigma), 50 nM phorbol myristate acetate (PMA, MP Biomedicals), or 400 ng/mL tumor necrosis factor alpha (TNFα, R&D Systems) followed by incubation for 20 hours at 37°C with 5% CO₂.
Human macrophages (8x10⁴ cells/ml) were seeded as above and incubated at 37°C with 5% CO₂ for 4 hours. After optimization of stimulant doses, cells received no treatment or were treated with 6.5 μM ionomycin (MP Biomedicals) or 25 ng/mL TNFα (R&D Systems) followed by the above incubation parameters. The following concentrations of MSU were tested: 1.2 mg/ml, 400 µg/ml, and 130 µg/ml.

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.