Siglec h percp cy5

Single-cell-suspensions of splenocytes and liver leukocytes were stained for cell surface markers using specific fluorophore-conjugated antibodies optimized for flow cytometry. Reagents and antibodies used in this study were LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher), CD11b-BUV395 (Clone: M1/70; BD Biosciences), Siglec-F-BUV615 (Clone: E50-2440; BD Biosciences), NK1.1-BUV661 (Clone: PK136; BD Biosciences), B220-BUV737 (Clone: RA3-6B2; BD Biosciences), CD8α-BUV805 (Clone: 53–6.7; BD Biosciences), MHCII(I-A/I-E)-BV510 (Clone: M5/114.15.2; BioLegend), CD4-BV570 (Clone: RM4-5; BioLegend), SCA-1-BV711 (Clone: D7; BioLegend), CD11c-BV785 (Clone: N418; BioLegend), Ly6C-FITC (Clone: HK1.4; BioLegend), Siglec-H-PerCP/Cy5.5 (Clone: 551; BioLegend), F4/80-PE (Clone: BM8; BioLegend), CD3ε-PE/Cy5 (Clone: 145-2C11; ThermoFisher), CD80-PE/Cy7 (Clone: 16-10A1; BioLegend), CD115-AF594 (Clone: AFS98; BioLegend), CCR2-APC (Clone: 475301; R&D Systems), CD45-AF700 (Clone: 30-F11; BioLegend), CD48-APC/Cy7 (Clone: HM48-1; BioLegend), Ly6G-Biotin (Clone: 1A8; BioLegend; with secondary antibody streptavidin-DyLight 800; ThermoFisher). Stained and fixed (4% PFA; 10 min in the dark) cells were analyzed with a Becton Dickson custom 10-laser ‘LSR-II’ flow cytometer and FlowJo software (v.10.4.1).

Isolation of total bone marrow cells and total splenocytes was performed as previously reported (12 (link), 19 (link)). Cell pellets were then suspended in 1 ml 1 × red blood cell (RBC) lysis buffer (eBioscience, San Diego, CA) and incubated for 5 min at room temperature, followed by neutralization of the lysis buffer with 5 ml of C10 medium. This solution was further centrifuged and the pellets were resuspended in 5 ml of fresh C10. Our C10 medium is complete RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, and 100 U/ml penicillin–streptomycin (all from Life Technologies, Grand Island, NY). The resulting mononuclear cells were stained for flow cytometry as we reported previously (12 (link)). For bone marrow dendritic cell analysis, the following anti-mouse monoclonal antibodies were used: CD11c-APC, CD11b-PE, CD11b-PErCp-Cy5, Siglec-H-PerCP-Cy5.5, I-E/I-A(MHC-II)-FITC (Biolegend, San Diego, CA), and Ly6C-APC-Cy7 (BD Biosciences, San Jose, CA). For analysis of splenic T-cell subsets, we used anti-mouse CD3-APC, CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD44-FITC, and CD62L-APC-Cy7 (Biolegend). Stained cells were analyzed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.