Rhm csf

Either 200ul PBS (control) or 20ug of recombinant MCSF (rhMCSF, Novartis) was injected into the peritoneums (i.p.) of wild type, C57/B6J 8–10 week old mice at indicated times prior analysis. For cell cycle analysis, mice were also injected i.p. with 2 mg of BrdU (BD Pharmingen) dissolved in PBS, as per manufacturers instructions 4h prior to analysis. To harvest peritoneal cells mice were sacrificed and subsequently injected with 10mL ice-cold PBS containing 2mM EDTA (Sigma). Intra-peritoneal washouts were collected in 50mL Falcon tubes. Red cell lysis was performed on total washouts (RBC Lysis Buffer, Invitrogen) prior to staining for cell surface markers and cell cycle analysis or FACS sorting. Remaining mononuclear cells were then stained using the following antibody cocktail in the presence of FcBlock (BD Biosciences): CD11b-PE-Cy7 (BD Biosciences), MHC-II-FITC, B220-APC-Cy7, F4/80-PE (all from eBioscience) and Fixable Aqua Dead-V500 (Life Technologies) and either sorted for single cell analysis or further processed for cell cycle analysis.