Anti mouse cd8 α ly 2 microbeads

For purification of MDSC subsets from tumor tissue, ear and tail skin of 4–5 late-stage tumor mice were digested with 0.15 mg ml⁻¹ Liberase (Roche) and 0.12 mg ml⁻¹ DNAse (Roche) for 45 min at 37 °C and pressed through 100-μm cell strainers (BD Biosciences). CD45⁺ cells were enriched with a 1.119 g ml⁻¹ Histodenz density gradient according to the manufacturer's protocol (Sigma-Aldrich, St. Louis, MO). The grMDSCs and moMDSCs were purified by flow cytometric cell sorting with a FACS Aria as follows: CD45⁺ CD11b⁺ cells were distinguished with Ly-6C and Gr-1 into Gr-1^inter Ly-6C^high moMDSCs and Gr-1^high Ly-6C^low grMDSCs. CD8⁺ T cells were isolated from LNs and spleen of pmel-1 mice before the onset of spontaneous vitiligo (2–3 months). Purification was performed with anti-mouse-CD8-α (Ly-2) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). In order to track T-cell proliferation, cells were stained with 0.4 μM CFSE for 5 min at room temperature. For suppression assays, 1 × 10² BMDCs loaded with 1 μM gp100 peptide (KVPRNQDWL, Peptide&Elephants) for 1 h at 37 °C. These cells were cultured with 2 × 10⁴ CFSE-labeled CD8⁺ T cells from pmel-1 mice and 2 × 10³ or 1 × 10⁴ sorted moMDSCs/grMDSCs for 3 days. T-cell proliferation was measured by analysis of CFSE dilution by flow cytometry.