Percp cy5.5 conjugated streptavidin

Cell suspensions from SPL, mLN, and cLP were analyzed by flow cytometry. Conventional CD4⁺ T cells and Tregs were stained with anti-CD3ε and anti-CD4 mAbs (BD Biosciences, San Jose, CA) and intracytoplasmic anti-foxp3 mAbs (eBioscience, San Diego, CA). Neutrophils were stained with Gr-1-FITC, CD11b-PECy7, and Ly6G-AF647 mAbs (eBioscience). Plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) were stained with MHCII-FITC, CD11b-PECy7, PDCA-1-AF647, and CD11c-APCCy7 mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin. The cDC subsets were stained with MHCII-FITC, CD11c-APCCy7, CD11b-PECy7, and CD103-PE mAbs with a lineage-negative mAb cocktail containing B220-biotin, CD3ε-biotin, Ly6G-biotin (eBioscience) and PerCP-Cy5.5-conjugated streptavidin (eBioscience). Monocytes and macrophages (MΦ) were stained with Gr-1-FITC, MHCII-PE, Ly6C-PerCP-Cy5.5, CD11b-PECy7, Ly6G-APC, and CD11c-APCCy7 mAbs (eBioscience). Blocking of FcγR binding was performed using mouse and rat serum (Jackson ImmunoResearch, West Grove, PA). Cells were analyzed on a FACS Canto II (BD Biosciences). Data were collected using FACS Diva software (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR).

Flow cytometry analysis was performed using anti-SSEA1-eFluor 660 (eBioscience 50-8813-42, 1:20), anti-CD44-FITC (IM7) (eBioscience, 11-0441-81, 0.5 mg per test) and anti-CD24-VioBlue (clone: 32D12) (Miltenyi biotech, 130-099-150, 1:11). For tumor-derived samples, anti-mouse MHC Class I H2-K^d (eBioscience 17-5957-82, 1:200) followed by PerCP-Cy5.5-conjugated streptavidin (eBioscience 45-4317-80, 1:200) was used to exclude host cells. Cells were incubated with antibodies at a concentration of 1 × 10⁶ cells/100 μL in sort buffer (15 mM HEPES buffer [Sigma H3375], 1% Bovine Serum Albumin [Sigma A9647], 2 mM EDTA [VWR 20302.260], 100 U/mL DNase I (NEB M0303), 100 U/mL penicillin and 100 μg/mL streptomycin [Thermo Fisher 15140122] in PBS) for 45 min on ice, washed 3 times with sort buffer and analyzed using LSR Fortessa (BD Biosciences) or sorted using FACSAria II (BD Biosciences). FACSDiva and FlowJo software were used to acquire and analyzed data, respectively. Gating strategy used for tumor analysis is shown in Supplementary Fig. 1.