Myeloid derived suppressor cell kit

Spleens and tumors were isolated from the mice and blood was drawn. Spleens were processed through a 70 µm cell strainer. Erythrocytes from spleen and blood were removed using the red blood cell lysis buffer (BD Biosciences, Heidelberg, Germany). Tumor tissue was minced into pieces and mechanically dissociated using the mouse Tumor Dissociation Kit with the gentleMACS™ Dissociator application (both Miltenyi Biotech, Bergisch Gladbach, Germany), according to the manufacturer´s instructions. The cell suspension was separated from tissue debris by sequentially using 100 µm and 70 µm cell strainers. For functional assays, untouched T cells were isolated using the Pan T cell isolation Kit II, and for MDSC isolation the Myeloid-Derived Suppressor Cell Kit was used (both Miltenyi Biotec). In brief, in a two-step separation process, PMN-MDSC (CD11b⁺ Ly6C^int Ly6G⁺) were isolated with anti-Ly6G beads followed by M-MDSC (CD11b⁺ Ly6C^high Ly6G⁻) isolation using anti-Gr1 beads. The purity of isolated cells was > 95% for T cells and between 75 and 90% for MDSC.
Cells from bone morrow cultures were isolated by FACSorting. Cells were stained as described for FACS analysis. FVD^negGr1^highMHC-II^low and FVD^negGr1^lowMHC-II^high were sorted on a BD Aria III system (BD Bioscience, Heidelberg, Germany).