Anti cdllb

TA muscles were dissected, dissociated mechanically, digested using 800U/ml collagenase II (10 ml per sample) for 1 h at 37°C, centrifuged and resuspended with lOOOU/ml collagenase II (1 ml per sample) and llU/ml Dispase (1 ml per sample) solution and incubated for 30 min at 37°C. The digested tissue was filtered using a 40 μm pore sized mesh and cells were centrifuged 5 min at 500 g. Cells were resuspended in FACS buffer (PBS containing 2% FBS and 2 mM EDTA), incubated for 15 min with Mouse BD Fc Block purified anti-mouse CD16/CD32 mAb (BD-pharmingen) and stained with the following antibodies for 30 min at 4°C: viability dye (Invitrogen), anti-CD45 (Biolegend), anti-CDllb (Invitrogen), anti-F4/80 (Invitrogen), anti-MHCII (Invitrogen), anti-CD80 (Invitrogen) and anti-CD206 (Biorad), anti-CD45R (BD Biosciences), anti-Ly6G (BD Biosciences,), anti-TCR(3 (BD Biosciences), anti-CD4 (BD Biosciences) and anti-CD8 (Invitrogen,). Cells were subsequently, washed and resuspended in cold FACS buffer before FACS analysis or flow sorting by a FACS Verse or FACS Aria (BD Biosciences), respectively. Fluorescence minus one (FMO) controls were performed in all the stainings and used for the proper gating in all the analysis.