Fcr blocker

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 Ly6G^highLy6C^{low or −}CD11b⁺, monocytic MDSCs as Ly6C^highLy6G^lowCD11b⁺. 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.

Single cell suspensions of tumor cells were incubated on ice with FcR blocker (eBioscience) in staining buffer (1% FBS in PBS), followed by cell surface marker staining with the following antibodies: CD24-PE, Epcam-APC, ScaI-PE, CD29-PECy7, CD49f-eFLUOR450 and cKit-APC and CD45-eFLUOR450 as indicated in figures. When intracellular staining was done, cells were fixed and permeabilized using reagents of eBioscience’s FOXP3 staining kit. Intracellular staining was performed in presence of 2% normal mouse serum, 2% normal goat serum and 2% FBS to reduce non-specific binding. Antibodies used were GCSF-APC (eBioscience) and rabbit anti-pS6K(T389, Cell Signaling), rabbit anti-pS6 (S235/236, Cell Signaling) or rabbit anti-pStat3(S727, Cell Signaling) followed by goat anti-rabbit-eFluor488.
For analyses of CSCs in tumor tissues, we first performed tumor dissociation using gentleMACS dissociators and tumor dissociation kit manufactured by Miltenyi Biotech. The cell suspension was then subjected to staining described above. Due to technical variations, tumors harvested together and analyzed at the same time were considered as an experimental set, and the systematic differences between different sets were removed by normalization (set the mean frequencies of “No treatment” samples as one within each set).