Ultrapure escherichia coli 0111 b4 lps free of lipoproteins

THP1 monocytes obtained from ATCC were differentiated into macrophage using 100 nM phorbol myristate acetate (PMA) for 5 h in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 % fetal bovine serum (FBS) (Sigma). Ultrapure Escherichia coli 0111:B4 LPS free of lipoproteins was obtained from Invitrogen (San Diego, CA).

Tamm-Horsfall protein 1 (THP-1) cells were maintained in RPMI-1640 supplemented with 2 mm l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA). Ultrapure Escherichia coli 0111:B4 LPS free of lipoproteins was obtained from Invitrogen (San Diego, CA, USA). The cells were plated in 22-mm tissue culture dishes (2×10⁶ cells/dish). Macrophage differentiation was induced with phorbol myristate acetate (PMA, 100 nM) for 5 h. In one set of experiments (pre-treatment experiments) the effects of H₂S were examined following a 30-min pre-treatment with sodium hydrosulfide (NaHS, an H₂S donor) (Sigma) at 0.01, 0.1, 0.5 or 1 mM followed by a washout, followed by incubation with bacterial lipopolysaccharide (LPS, 1 μg/ml) in 1% FSB RPMI-1640 for 1, 4, 8 or 24 h. In another set of experiments (co-treatment experiments), NaHS was administered 30 min prior to the LPS administration, without a washout. The control cells were maintained in 1% FBS RPMI-1640.