Cd206 clone c068c2

A single‐cell suspension was derived from the BALF of LysMCre:Shp2^fl/fl and Shp2^fl/fl mice 24 hour after secondary S aureus infection. After blocking the Fc receptor, cells were stained with CD11b (clone M1/70, BioLegend, San Diego), F4/80 (clone BM8, BioLegend) and Dectin‐1 (clone RH1, BioLegend), or their corresponding IgG isotype controls. After extracellular markers were stained, the cells were fixed, permeabilized and stained with CD206 (clone C068C2, BioLegend) or its isotype control. Cell samples were detected by CytoFLEX flow cytometer (Beckman Coulter, Inc.), and FlowJo software (TreeStar, Inc.) was used for analysis.

After in vivo experiments, mouse tumors were rapidly excised and mechanically dissociated in PBS using scissors. The tumors were then digested in serum-free RPMI supplemented with 20 mg/ml DNase I (Roche), 20 mg/ml Dispase II (Roche) and 20 mg/ml collagenase I (Sigma) for 30–60 min at 37 °C with rotation to promote dissociation. The resulting single-cell suspensions were passed through 70 μM strainers twice, and red blood cells in the tumor samples were lysed with red blood cell lysis buffer (eBioscience, #00-4333-57) for 5 min at room temperature. Then, the single-cell suspensions were washed in Cell Staining Buffer (BioLegend) and incubated with the indicated flow antibodies at 4 °C for 30 min. Prior to staining with antibody panels, cells were blocked with a monoclonal antibody against CD16/32 (BioLegend) for 15 min at 4 °C. All the antibodies and reagents used for flow cytometry included ZombieRED (ECD, Biolegend, #423110), CD45 (clone 30-F11, Biolegend, #103116), CD3e (clone 145-2C11, Biolegend, #100328), CD4 (clone RM4-5, Biolegend, #100536), CD8 (clone 53–6.7, Biolegend, #100706), GZMB (clone QA16A02, Biolegend, #372208), PRF1 (clone S16009B, Biolegend, #154404), F4/80 (clone BM8, Biolegend, #123127), CD11b (clone M1/70, Biolegend, #101206), CD86 (clone GL-1, Biolegend, #105006) and CD206 (clone C068C2, Biolegend, #141719).

Single-cell suspensions from muscle were prepared by collagenase II (Worthington Biochemical Corp, Lakewood, NJ, USA) digestion followed by staining with fluorochrome-conjugated antibodies.
Antibodies used: (1) Gr1 Clone RB6-8C5 (eBioscience, ThermoFisher, Waltham, MA, USA, Cat#48-5931-82); (2) F4/80 Clone BM8 (Invitrogen, Cat# 11-4801-82); (3) CD11b Clone M1/70 (Invitrogen, ThermoFisher, Waltham, MA, USA, Cat#17-0112-81); (4) Siglec-F Clone E50-2440 (RUO), (BD BioSciences, ThermoFisher, Waltham, MA, USA, Cat# 552126); (5) CD206 Clone C068C2 (BioLegend, San Diego, CA, USA, Cat#141701) and live dead stain (eBioscience). Data acquisition was performed on a FACS Aria or Fortessa (BD Biosciences). UltraComp eBeads (Cat#01333342, Invitrogen) were used to generate single-stain controls. Data was analyzed using FlowJo software (version 10.2) and gating strategies as previously described [39 (link)] to discriminate against dead cells, debris, and doublets were utilized. M1 macrophages were defined as Gr1^low-med, F4/80⁺, and CD11b⁺.

Tumor tissues were processed into single-cell suspensions by mincing, as well as chemical (Murine Tumor Dissociation Kit, Miltenyi Biotec) and mechanical (gentleMACS Dissociator) dissociation, per manufacturer recommendations. Suspensions were filtered through a 100 μm filter and washed with 1% BSA in PBS prior to blocking nonspecific staining with anti-CD16/32 (BioLegend) antibody. Cell surface staining was performed using fluorophore-conjugated anti–mouse CD45.2 (clone 104), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD31 (clone 390), PDGFR (clone APA5), PD-L1 (clone 10F.9G2), H2-K^b (clone AF6-88.5), CD107a (clone 1D4B), PD-1 (clone RMP1-30), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone HK1.4), CTLA-4 (clone 9H10), CD39 (clone Duha59), CD11c (clone N418), F4/80 (clone BM8), and CD206 (clone C068C2) from BioLegend, and IL-12Rb2 (clone 305719) from R&D Systems. FoxP3⁺ Treg staining performed with the mouse Treg Staining Kit #1 (eBioscience) as per manufacturer’s protocol. Cell viability was assessed via staining with Sytox (Thermo Fisher Scientific) or Zombie (BioLegend) dyes. All analyses were performed on a BD Fortessa analyzer running FACSDiva software and interpreted using FlowJo V.X10.0.7r2.