Cell isolation and culture

CSF-2 BMDM and BMDC and CSF-1 BMDM were prepared as described previously^27,28. Briefly, bone marrow cells were isolated from the femurs and tibias of mice and treated with RBC lysis buffer (0.84% ammonium chloride, 2 mM Tris-Cl pH 7.2) for 5 min at RT. Two million bone marrow cells were cultured in a 100 × 15-mm petri dish (Falcon) in 10 ml complete RPMI 1640 medium (10% fetal bovine serum (FBS) 20 mM Hepes, 1× nonessential amino acid, 55 μM 2-mercaptoethanol, 50 U/ml penicillin/streptomycin, 1 mM sodium pyruvate (all from Invitrogen), 2 mM L-glutamine, (Sigma-Aldrich) supplemented with J558L supernatant containing 20 ng/ml of CSF-2). Non-adherent cells were harvested on day 7. For CSF-1 derived BMDMs, 1.5 × 10⁷ bone marrow cells were cultured in 10 ml of DMEM, 20% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 U/ml penicillin/streptomycin, and 8% L929 cell-conditioned media (LCCM) as a source of CSF-1. BMDMs were harvested using Versene at day 5 and re-plated in a 96-well plate overnight, and cells stimulated on day 6. Peritoneal macrophages were obtained by peritoneal lavage using Ca²⁺ and Mg²⁺ free Hanks’ balanced salt solution (Invitrogen). Recovered cells were plated in DMEM with 20% FBS in a flat bottom 96-well plate for 2 hr, non-adherent cells discarded, and the remaining adherent cells (enriched for peritoneal macrophages) used for analysis. Splenic cells were isolated from spleen homogenized by collagenase digestion (1 mg/ml collagenase IV (Worthington) in PBS and 5% FBS) for 20 min at RT.