Cd49b pe dx5

Peritoneal cells were isolated by washing the peritoneal cavity twice with cold PBS 1x. Peritoneal exudate were then treated with ACK Lysis buffer to lyse red blood cells and washed once with PBS. Cells were then re-suspended to single-cell suspensions for staining with fluorescently conjugated antibodies at 1:100 dilutions, unless otherwise noted. Antibodies were diluted using 2% fetal bovine serum (FBS). Cells were stained with one of either LIVE/DEAD™ Blue (Invitrogen) or LIVE/DEAD™ Near-IR (Invitrogen), blocked with 4~g/ml anti-CD16/32 (2.4G2; Bioxcell) and stained with anti-CD11b Pacific Blue (M1/70; Biolegend), F4/80 PECy7 (BM8; Biolegend), CD206 APC (C068C2; Biolegend), Siglec-F PE (E50–2440; BD Biosciences), CD3 PE (145–2C11; Biolegend), CD19 PE (6D5; Biolegend), CD49b PE (DX5; Biolegend), Ly6G (1A8; Biolegend), PD-L2 (PerCP-Cy55; Miltenyi; diluted at 1:20), MHCII (APC-Cy7; Biolegend). Cells were gated on singlet, live, Dump-negative (CD3–, CD19–, DX5–, Siglec-F–, Ly6G–), CD11b+, then subsequently gated on their M^res and AAM^res (F4/80hi, CD206–) or M^mono and AAM^mono (F4/80int, CD206+) phenotype. Cell surface expression of PD-L2 and MHCII were acquired for analysis. Cells were sorted using 100μm nozzle into FBS, on either BD FACSAriaII or SONY HAPS1, depending on instrument availability.

Cells were labeled for flow cytometry by incubation with the following fluorophore-conjugated antibodies: CD3 perCP-Cy5.5(17A2; Biolegend), Cd49b PE (DX5; Biolegend), NKp46 APC (29A1.4; Biolegend), CD11b FITC (M1/70; eBioscience), Ly6G PE (1A8; BD Pharmingen), G-CSF Receptor (S1390; Abcam), CD107a (1D4B; Biolegend). Flow cytometric analyses were carried out on a fluorescent -activated cell sorting (FACS) Fortessa (BD Biosciences) using BD FACSDiva Software.