BMDM differentiation and stimulation

BMDMs were generated by plating mouse BM cells in non–tissue culture (TC) dishes in macrophage growth medium (MGM) composed of 30% L929 supernatant/70% RPMI 1640 with l-glutamine (Corning). RPMI 1640 was supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), penicillin/streptomycin, sodium pyruvate, Hepes, and 2-mercaptoethanol. BM was prepared by crushing mouse femurs and tibias to release marrow, followed by ACK (ammonium-chloride-potassium) lysis and passage through a 70-μm cell strainer. BM was plated on day of isolation (day 0) at 7 × 10⁶ cells/20 ml of MGM in a 15-cm non-TC dish. On day 4 of differentiation, cells were supplemented with 10 ml of MGM. Unless otherwise stated, on day 6, adherent cells were lifted with cold phosphate-buffered saline (PBS) containing 5 mM EDTA and were replated at 0.75 × 10⁶ cells per well of 12-well non-TC dish or 1.5 × 10⁶ cells per well of six-well non-TC dish in MGM for stimulation on day 7 of differentiation. All cell culture was done at 37°C with 5% CO₂. LPS stimulation was performed with Ultrapure LPS, Escherichia coli 0111:B4 (Invivogen, catalog no. tlrl-3pelps). Concentrations and length of stimulations are indicated in the figure legends and/or the results section of the main text. If intracellular cytokine staining was to be performed, GolgiPlug (BD Biosciences, catalog no. 555029), a BFA-containing reagent, was added at the time of stimulation.