Cd8 isolation kits

Murine cells were obtained from adult mouse lymph nodes and spleens. Obtained cells were treated with ACK lysing buffer to lyse red blood cells and filtered through cell strainers to isolate splenocytes. PBMCs from healthy donors were isolated by Ficoll-Paque PLUS gradient centrifugation (GE Healthcare). CD8+ T lymphocytes were then isolated from splenocytes or PBMCs by negative selection using CD8+ isolation kits and magnetic columns from Miltenyi Biotech (Auburn, CA, USA) according to the manufacturer’s protocol. PBMCs were obtained from blood drawn from healthy males and females per JHU IRB approved protocols.

We adoptively transferred 10⁶ Teffs, 10⁶ CD4s and either no Tregs or 10⁵ Tregs (WT, CXCR3^−/− or CCR6^−/− Tregs) into 8-12-week-old Rag^−/− hosts infected i.p. with 10⁶ PFU of rVV-hPMEL on the same day of adoptive transfer. Hosts were irradiated with 500 rads 1 day before transfer. In competitive Rag^−/− vitiligo models, vitiligo was induced by adoptive transfer of 10⁶ Thy1.1+ Teffs, 10⁶ CD45.1+ CD4+ T cells, and 5.0×10⁴ Thy1.1+Thy1.2+ WT and 5.0×10⁴ Thy1.2+ KO Tregs (CXCR3^−/− or CCR6^−/−) into 8-12-week-old Rag^−/− hosts irradiated with 500 rads 1 day before transfer, and hosts were infected i.p. with 10⁶ PFU of rVV-hPMEL on the same day of adoptive transfer. Teffs were isolated using a Miltenyi Biotec CD8 isolation kits. Treg-depleted CD4+ T cells were isolated from the spleens of C57BL/6J or CD45.1 mice through negative selection using Miltenyi Biotec CD4+CD25+ isolation kits (Bergisch Gladbach, Germany). Tregs were isolated from the spleens of C57BL/6J mice, CXCR3^−/− mice, or CCR6^−/− mice through positive selection using Miltenyi Biotec CD4+CD25+ isolation kits.