Gsk2033

Monocytes were primed by culturing 40.000 cells/well in a 96-well plate (Greiner Bio-One^TM) with 2 μg/ml BCG, 2 μM T0901317 (T1317) (Cayman, #71810), 10 μM GW3965 (Cayman, #10054), 5 μM GSK2033 (Cayman, #25443), 500 μM acetyl-CoA (Cayman, # 16160) or 10 ng/ml IL-1β (Peprotech, #200-01A) for 24 h in RPMI supplemented with 10% pooled human AB serum, 5 mM glucose and 1% Penicillin/Streptomycin. In experiments in which inhibitors were used, cells were pre-incubated for at least an hour with 100 nm Torin1 (Cayman, #10997), 20 μM MTA (Cayman, #15593), 10 μM LW6 (Axonmedchem, # 2480), 10 μM KC7F2 (Tocris, #4324), 20 μM Fluvastatin (Sigma, SML0038), 25 μM C75 (Tocris, #2489), 1 μM diacerein (Cayman,# 11710), 200 ng/ml IL-1RA (Peprotech, #200-01RA) prior to priming. The medium was changed after 24 h and cells were rested for 5 days or as indicated. 50% of the medium was refreshed on day 3. On day 6 medium was changed and cells were re-stimulated with either 200 μL RPMI containing 5 μg/ml of Pam3Cys (EMC mirocollection, #L2000), 10 ng/ml of LPS (Sigma) or with medium only. DMSO control was always included when there was a compound dissolved in DMSO. All working solutions were prepared in culture media. The plates were incubated in a 5% CO2 incubator maintained at 37°C. After 24 h supernatants were collected and stored at −20°C until used for cytokine assay.