Purified human CD8
natural Tregs express FOXP3 and CTLA-4. Separated cell fractions, CD8+CD25+ and CD8+CD25− T cells, were analyzed by multicolor FACS using the following antibodies: anti-CD3 PerCP, anti-CD8-APC (BD biosciences) and anti-CD25-PE (Miltenyi Biotec) to assess purity for each isolation. Intracellular FOXP3 and cell-surface CTLA-4 were assessed using human anti-FOXP3-PE detection kit and anti-CTLA-4-PE (BD biosciences). Flow cytometry was performed using a FACSCalibur applying CellQuest software (BD Biosciences). CD3 and CD8 purity was above 97% among isolated CD8+ T cells. CD8+CD25+ nTregs express FOXP3 and CTLA-4 when compared to CD8+CD25− T cells.