Inflammasome Activation in Macrophages

BMDM were seeded at 5 ×₀ 10⁵/ml or 1 × 10⁶/ml, HMDM at 5 × 10⁵/ml and PBMC at 2 × 10⁶/ml or 5 ×₀ 10⁶/ml in 96 well plates. The following day the overnight medium was replaced and cells were stimulated with 10 ng/ml LPS from Escherichia coli serotype EH100 (ra) TLRgrade™ (Alexis Biochemicals) for 3 h. Medium was removed and replaced with serum free medium (SFM) containing DMSO (1:1,000), MCC950 (0.001–10 µM), glyburide (200 µM) (Sigma Aldrich), parthenolide (10 µM) (Enzo Life Sciences) or Bayer cysteinyl leukotriene receptor antagonist 1-(5-carboxy-2{3-[4-(3-cyclohexylpropoxy)phenyl]propoxy}benzoyl)piperidine-4-carboxylic acid (40 µM) (Amgen Inc., Thousand Oaks, CA, USA) for 30 min. Cells were then stimulated with inflammasome activators: 5 mM adenosine 5’-triphosphate disodium salt hydrate (ATP) (1 h), 1 µg/ml Poly(deoxyadenylic-thymidylic) acid sodium salt (Poly dA:dT) (Sigma Aldrich) transfected with Lipofectamine 2000™ (Invitrogen) (3–4 h), 200 µg/ml MSU (overnight) and 10 µM nigericin (Invivogen) (1 h) or S. typhimurium UK-1 strain (M.O.I. 20) obtained from Dr. Sinead Corr, Trinity College Dublin, Ireland (2 h). Cells were also stimulated with 25 µg/ml Polyadenylic-polyuridylic acid (Invivogen) (4 h). For non-canonical inflammasome activation cells were primed with 100 ng/ml Pam3CSK4 (Invivogen) for 4 h, medium was removed and replaced with SFM containing DMSO or MCC950 and 2 µg/ml LPS was transfected using 0.25% FuGENE® (Promega) for 16 h. Supernatants were removed and analysed using ELISA kits according to the manufacturer’s instructions (DuoSet® R&D Systems or ReadySetGo!® eBioscience). LDH release was measured using the CytoTox96® non-radioactive cytotoxicity assay (Promega)