Be17 605f

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque PLUS (GE Healthcare, GE17-1440-03) density centrifugation of whole blood from healthy donors (National Research Ethics Service reference 07/Q2305/7). PBMC were seeded in RMPI 1640 media (Gibco, 11875-093) containing 2 mmol/L L-glutamine (Lonza, BE17-605F) and 10% newborn calf serum (Gibco, 26010074). Cells were cultured in 5% CO₂ at 37°C for 14 days in RPMI 1640 supplemented with 2 mmol/L L-glutamine and 10% heat-inactivated fetal bovine serum (FBS) (PAN Biotech, P30-3306) (38 (link)).
RPMI 1640 + 25 mM HEPES (Gibco, 11550496) containing 2 mmol/L L-glutamine and 10% heat-inactivated FBS and drugs were pre-equilibrated in normoxia or hypoxia (0.8% O₂, 5% CO₂, 70% humidity at 37°C, in a Baker-Ruskinn Sci-Tive UM-027) for 24 h prior to the experiment (39 (link)). Cells were treated in duplicate or triplicate wells with 100 μg/ml LPS (InvivoGen, tlrl-b5lps), DMSO (Sigma-Aldrich, D8418), or 15 mM COX-2 inhibitor (NS398) (Cayman Chemical, 70590) and incubated for 18 h. For normoxic controls, cells were treated identically within a class-2 tissue culture hood and transferred to a normoxic tissue-culture incubator.
Cell supernatants were collected following 18 h incubation and were assayed in triplicate using a human TNF ELISA MAX (Biolegend, 430204).