Pulmonary macrophage isolation

Pulmonary macrophages were isolated from naïve mouse lungs as previous described [15]. Briefly, cells were mechanically isolated from mouse lung tissues through 70um cell strainers. Macrophages then were purified by gradient centrifugation (Histopaque 1083; Sigma-Aldrich) and seeded at a concentration of 1×10⁶ cells/well in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich) containing 20% fetal bovine serum (FCS). After 3h, all non-adherent cells were removed and >95% of adherent cells were macrophages. Isolated macrophages were then stimulated with PBS or LPS (50ng/ml; Sigma-Aldrich) and IFNγ (1.5μg/ml; Pepro Tech) in the presence of I-BET-762 (10μM; Merck Millipore) or vehicle (20% hydroxyl-β cyclodextrin, 2% DMSO in 0.9% saline) (Sigma, Aldrich) for 12h, then harvested into Trizol for mRNA qualification.