Ultrapure LPS was purchased from Invitrogen and used at a concentration of 10 μg/mL. selleck compound ATP was from Sigma and used at a final concentration of 3 mM. For immunoblotting, cells were washed twice with sterile PBS and lyzed in buffer (150 mM NaCl, 10 mM Tris, pH

7.4, 5 mM EDTA, 1 mM EGTA, 0.1% Nonidet P-40) which was supplemented with a Roche protease inhibitor cocktail tablet. After clarification and denaturation with SDS buffer, samples were boiled for 5 min. Separation of the proteins was done by using SDS-PAGE and thereafter transferred into a nitrocellulose membrane. These membranes were coated with primary antibodies and active caspase-1 was detected using secondary anti-rabbit antibody conjugated to horseradish peroxidase followed by enhanced chemiluminiscence. Sotrastaurin manufacturer Peritoneal macrophages were isolated by injecting

5 mL of ice-cold sterile PBS (pH 7.4) in the peritoneal cavity. After centrifugation and washing, cells were resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM L-glutamine and 100 μg/mL gentamycin (culture medium). Cells were counted using a Z1 Coulter Particle Counter (Beckman Coulter, Woerden, The Netherlands) and adjusted to 1×106 cells/mL. Cells were cultured in 96-well round-bottom microtiter plates (Costar, Corning, The Netherlands) at 1×105 cells/well, at a final volume of 200 μL. The cells were stimulated with RPMI or two different heat-killed Borrelia strains. After 24 h of incubation at 37°C in air and 5% CO2, the plates were centrifuged at 500×g for 10 min, and the supernatant was collected and stored at −80°C until cytokine assays were performed. Investigating the role of IL-33 was not performed by incubation of peritoneal macrophages that were stimulated with RPMI or Borrelia in the presence or absence of 10 μg/mL anti-mouse

IL-33 antibody (R&D Systems). After 48 h of incubation, IL-4 and IL-5 levels were measured using ELISA kits (eBioscience, San Diego, CA, USA). ELISA were performed according to the manufacturer’s instructions. Spleen cells were isolated by gently squeezing spleens in a sterile 200 μm filter chamber. After washing with sterile PBS and centrifugation at 4°C (1200 rpm, 5 min), cells were resuspended in 4 mL RPMI 1640 in presence of 20% FCS. Cells were counted and concentrations were adjusted to 1×107 cells/mL. Cells were cultured in 24-well plates (Greiner, Alphen a/d Rijn, The Netherlands) at 5×106 cells/well, at a final volume of 1000 μL. After 5 days of incubation, supernatant was collected and stored at −80°C until cytokine assays were performed. Concentrations of mouse IL-1β were determined by specific radioimmunoassay (RIA; detection limit is 20 pg/mL) as described by Netea et al52. Mouse IL-6, IL-17 and IFN-γ, concentrations were measured by a commercial ELISA kit (Biosource, Camarillo, CA; detection limits 16 pg/mL), according to the instructions of the manufacturer.