Mouse bone marrow-derived macrophages

Femurs and tibias of mice were collected in RPMI (ThermoFisher, 61870036), surface sterilized with ethanol and flushed with cold HBSS (ThermoFisher, 24020117) using a 25 g needle and syringe. Clumps and fragments were removed by aspirating up and down with the syringe followed by filtering through a 40 μM strainer into a 50 ml tube. Bone marrow cells were centrifuged at 300g for 5 min, resuspended and counted in cold RPMI. Cells were adjusted to concentration in RPMI containing 10% heat-inactivated FBS (Serana, S-FBS-SA-015), 100U/ml penicillin (Eureco-pharma), 100 μg/ml streptomycin (Sigma, S9137), 50 μM 2-mercaptoethanol (Sigma, M3148) and 10% supernatant of M-CSF producing L929 cells. 5 ml containing 2 × 10⁶ cells were plated in non-culture treated 6-well plates. Cultures were supplemented on day 3 or 4 with 5 ml of complete RPMI containing 20% L929 supernatant. Cells were harvested using accutase (Sigma, A6964) on day 7, replated in 24-well plates with (3–5) × 10⁵ cells/well and stimulated in 1 ml for 18–24 h with LPS (100 ng/ml) and IFNy (50 ng/ml, BioLegend, 575304) or IL-4 (20 ng/ml, BioLegend, 574304).