To characterize and quantify MDSCs by FACS, cells were incubated with FcR blocker (eBioscience), then stained with Ly6C-PE-CF594 (BD Biosciences), Ly6G-PerCPCy5.5 (BD Bioscience), CD11b-APC (eBioscience) and/or anti-GR1 (RB6, BD Biosciences). MDSCs were identified as GR1+CD11b+ cells, granulocytic MDSCs as Ly6GhighLy6Clow or −CD11b+, monocytic MDSCs as Ly6ChighLy6GlowCD11b+. To determine absolute cell numbers counting beads (BD Biosciences) were added before FACS acquisition. T-cells were analyzed with an antibody combination of CD3-PerCPCy5.5, CD4-APC, CD8-FITC (all eBioscience) and PD1-PE-CF594 (BD Biosciences). Please also see Supplementary Table 2 for antibody use information.
Tumor infiltrating MDSCs and T-cells were quantitated by enzymatic digestion of the dissected tumors (Mouse tumor cell dissociation kit, Miltenyi Biotec), followed by FACS staining with the above antibody combinations. Staining for the common leukocyte marker CD45 was also included to facilitate the distinction of leukocytes from tumor cells and other stromal cells.
Tumor infiltrating MDSCs and T-cells were quantitated by enzymatic digestion of the dissected tumors (Mouse tumor cell dissociation kit, Miltenyi Biotec), followed by FACS staining with the above antibody combinations. Staining for the common leukocyte marker CD45 was also included to facilitate the distinction of leukocytes from tumor cells and other stromal cells.