F4 80 pe cy7 bm8

Single cell suspensions were stained with Live/Dead Yellow (Life Technologies, Grand Island, NY), CD11b-PerCp/Cy5.5 (M1/70, Biolegend, San Diego, CA), I-A/I-E-AF488 (M5/114.15.2, Biolegend), Gr-1-Pacific Orange (RB6-8C5, Life Technologies), F4/80-PE-Cy7 (BM8, Biolegend), CD11c-APC-Cy7 (HL3, BD Biosciences, San Jose, CA), CD117-PE-Cy5 (2B8, Biolegend) and FcεRI (Mar-1, Biolegend). Ly6-C-Pacific Blue (HK1.4, Biolegend) and Ly6-G-AF647 (1A8, Biolegend) were used in adoptive transfer experiments. For intracellular cytokine staining, cells were stimulated for 4.5 h with 30 nM phorbol 12-myristate 13-acetate and 1 μM ionomycin (both eBioscience) in presence of Golgistop (BD). Cells were subsequently stained against cell surface markers (CD4-PerCp/Cy5.5, GK1.5; CD3-AF700, 17A2; CD8-PE/CF594, 53-6.7; all Biolegend) followed by fixation/permeabilization (Cytofix/Cytoperm, BD) and staining using IL-17A-Pacific Blue (TC11-18H10.1, Biolegend). Flow cytometry was performed using a LSRII cytometer (BD) and data were analyzed with FACSDi-Va 6.1.3 software (BD). For cell sorting (FACS) experiments, myeloid populations were sorted using an AriaII cytometer (BD). MO-MDSCs were sorted as CD45⁺CD3^-CD11b^hiGr-1^lowF4/80^-/lowMHC-II^-/low, PMN-MDSCs as CD45⁺CD3^-CD11b^hiGr-1^hiF4/80^-MHC-II^-, and MΦ as CD45⁺CD3^-CD11b^hiGr-1^-/lowF4/80^hiMHC-II⁺ cells.

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.

Peritoneal lavage fluid was stained with F4/80-PE/Cy7 (BM8) (Biolegend) and CD11b-APC/A700 (M1I70) (eBioscience) antibodies for 35 min. Red blood cells (RBCs) were lysed with RBC lysis buffer (Becton Dickinson) for 5 min. Cells were fixed with 1% paraformaldehyde and analysed on an LSRII flow cytometer (BD). Macrophages were defined as F4/80+CD11b+ cells.