Bone marrow was harvested from the femur and tibia of mice by flushing with sterile PBS as previously described (3 (link), 4 (link)). Cells were washed twice in PBS and filtered through a 70-μm sieve. The single-cell suspension was then cultured in macrophage media (DMEM/F12 supplemented with 10% FCS and L929-conditioned media). To fully differentiate BMDMs, cells were cultured for 7 days with fresh media changed every 3 days. Adherent macrophages were detached from plates using a cell scraper, and cell viability was assessed by trypan blue exclusion.
For Western blot analysis, BMDMs were seeded onto six-well plates at a density of 1 × 106 live cells per well in fresh macrophage media. Where indicated, BMDMs were either unstimulated or stimulated the next day with LPS (100 ng/ml; Sigma-Aldrich, #L2630) or murine recombinant IL-4 (20 ng/ml; Peprotech, #214-14). At the indicated time points, cells were washed with ice-cold PBS, detached from plates using a cell scraper, and processed for Western blot analysis.
For Western blot analysis, BMDMs were seeded onto six-well plates at a density of 1 × 106 live cells per well in fresh macrophage media. Where indicated, BMDMs were either unstimulated or stimulated the next day with LPS (100 ng/ml; Sigma-Aldrich, #L2630) or murine recombinant IL-4 (20 ng/ml; Peprotech, #214-14). At the indicated time points, cells were washed with ice-cold PBS, detached from plates using a cell scraper, and processed for Western blot analysis.
