Lps e coli strain o111 b4

Poly-d-lysine, cytosine β-d-arabinofuranoside (Ara-c), l-leucine methyl ester (LME) and 3,3′-diaminobenzidine, mazindol, and urea-hydrogen peroxide tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS (E. coli strain O111:B4) and NADPH oxidase inhibitor DPI were purchased from Sigma (San Diego, CA, USA). Cell culture ingredients were obtained from Life Technologies (Grand Island, NY, USA). Antibody diluents were purchased from DAKO (Carpinteria, CA, USA). Anti-complement C3 antibody, Complement C3, and iC3b protein (endotoxin-free) were purchased from Millipore Sigma, and the anti-Iba1 antibody was purchased from Wako Pure Chemicals (Richmond, VA, USA). Anti-3-nitrotyrosine (3-NT) and anti-tyrosine hydroxylase (TH) antibodies were purchased from Abcam (Cambridge, MA, USA). TNF-α ELISA kit and isotype control antibodies were purchased from R&D Systems (Minneapolis, MN, USA). Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-goat IgG were purchased from cell signaling (Burlingame, CA, USA).

MDMs were challenged with 1 µg/ml bacterial lipopolysaccharide (LPS, E. coli strain O111:B4; Sigma-Aldrich, St. Louis, MO) for 4 h with subsequent addition of 1 mM ATP (Sigma-Aldrich, St. Louis, MO) for 15 min or cytoplasmic delivery of 5 µg/ml flagellin from S. typhimurium (FLA, InvivoGen, San Diego, CA) in DOTAP liposomes (6 µg DOTAP (Roche)/1 µg FLA) for 2 h before supernatants were collected. Long-term stimulation was performed with 1 µg/ml LPS or 5 µg/ml FLA for 24 h.