Macsquant10 flow cytometry

Tumor suspensions were obtained after mechanical and enzymatic disaggregation (Accumax (Merck Millipore, Burlington, MA, USA) (15 min, room temperature (RT)) and filtered through 70 μM nylon mesh cell strainer (Thermo Fisher Scientific). Erythrocytes were lysed with Quicklysis buffer (Cytognos, Salamanca, Spain) and cells were incubated with hFcR Blocking (Miltenyi Biotec, Bergisch Gladbach, Germany), previous to antibody (Table S2) incubation (20 min at 4 °C in PBS 1% fetal bovine serum (FBS)). Cells were labelled with a Fixable Viability Stain (Becton Dickinson, Franklin Lakes, NJ, USA) (20 min, RT). The analysis was conducted in a Macsquant10 flow Cytometry (Miltenyi Biotec). Subset definition was: Neutrophils: CD45⁺CD11b⁺CD16⁺CD15⁺CD14⁻CD33⁻; myeloid-derived suppressor cells (MDSCs): CD45⁺CD11b⁺CD16⁺CD15⁻CD14^+/−CD33⁺; Macrophages: CD45⁺CD11b⁺CD16⁻CD15⁻CD14^+/−CD33⁻MHCII⁺; Tregs: CD45⁺CD3⁺CD4⁺CD25⁺CD127^lo.