CD8+ T cells enrichment from PBMC and sorting of CD8 Treg
CD8 T cells were purified from PBMC with magnetic beads using EasySepTM Human CD8 Positive Selection Kit II according to manufacturer’s protocol. The purity of CD8+ T cells was more than 94%. Enriched CD8 cells were activated with 15μl/ml of human T-Activator CD3/CD28 in RPMI medium for 48 hrs. Cells were washed and stained with monoclonal antibodies defining two different types of CD8 Treg, CD8 PerCP cy 5.5, CD183 PE, CCR7 FITC and CD45RA APC and CD8 PerCP cy 5.5, CD183 PE, CD25FITC and CD278 (ICOS) Alexa Fluor 647 for 20min. Cells were washed and sorted for CD8+/CD183+/CD25High+/CD278+ and for CD8+/CD183+/CCR7+/CD45RA- by BD FACS Aria II Cell Sorter (Becton Dickinson, San Jose, California).