This suggests that ATP does not sufficiently activate P2X7R in swine macrophages cultured

in vitro. ATP, BzATP, lipopolysaccharide (LPS), LPC (1-palmitoyl-sn-glycero-3-phosphocholine), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). A438079 was purchased from Tocris (Bristol, UK). Biotinylated anti-mouse IL-1β (BAF401) and anti-swine IL-1β (BAF681) antibodies were purchased CHIR-99021 datasheet from R&D Systems (Minneapolis, MN). Horseradish peroxidase (HRP)–streptavidin conjugate was purchased from Zymed (South San Francisco, CA). Anti-P2X7R rabbit polyclonal (Epitope: KIRKEFPKTQGQYSGFKYPY from the C-terminus of rat P2X7R, mouse-18/20 and swine-15/20 residues identical) and goat polyclonal (Epitope: YETNKVTRIQSMNY from the N-terminus of human P2X7R, mouse-13/14 and swine-14/14 residues identical) antibodies were purchased from Alomone Labs (Jerusalem, Israel) and Covalab (Villeurbanne, France), respectively. Selleckchem Ku0059436 Anti-actin mouse monoclonal antibody was

purchased from Chemicon International (Temecula, CA). HRP-conjugated rabbit anti-goat, goat anti-rabbit, and goat anti-mouse immunoglobulins antibodies were purchased from ICN Pharmaceutical, Inc. (Aurora, OH). Swine neonates (1–14-days-old crossbred pigs) were obtained from the animal facility at the National Institute of Livestock and Grassland Science, according to the institutional guidelines for animal experiments. After anesthesia had been induced and the animals had been euthanized, their kidneys were dissected out. After the removal of the fibrous renal capsule, the renal cortex was cut into small pieces, and the tissue pieces (3–5 g)

were then forced through a nylon mesh (pore size: 500 µm) in phosphate-buffered saline (PBS) using a scraper. The minced tissue was digested by incubation with collagenase–dispase (Roche Diagnostics, Basel, Switzerland)/PBS solution (1 mg/ml) containing DNase I (Roche) (40 µg/ml) for 1 h at 37 °C. Then, the digested tissue fragments were collected and re-suspended in growth medium composed of Dulbecco’s modified Eagle’s medium (Sigma) containing 10% heat-inactivated Vasopressin Receptor fetal bovine serum (Sanko Junyaku Co., Ltd., Tokyo, Japan), supplemented with 100 µM β-mercaptoethanol (Sigma), 10 µg/ml insulin (Sigma), 100 µg/ml streptomycin (Life Technologies, Carlsbad, CA), 100 U/ml penicillin (Life Technologies), and 5 µg/ml Fungin (InvivoGen, San Diego, CA). The cell suspension was split into 10 T-75 tissue culture flasks (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and cultured at 37 °C in a humidified atmosphere of 95% air/5% CO2. The culture medium was replaced every 3–4 days. After the cells had been cultured for 2–3 weeks, the cultured cells were harvested by treatment with Accumax™ (Innovative Cell Technologies, Inc., San Diego, CA), suspended in Cell Banker 1 (Nippon Zenyaku Kogyo Co., Ltd.