Isolation and FACS Sorting of Tregs and Teffs

Tregs and CD4⁺ Teffs were freshly isolated from buffy coats obtained from 13 volunteer blood donors according to our previously described protocol. Briefly, on the day of blood donation peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll/Uropoline gradient centrifugation and subjected to negative immunomagnetic selection (StemCell Technologies, Canada). Subsequently, CD4⁺ T cells were stained with monoclonal antibodies (mAb) specific for the following antigens: CD3, CD4, CD25, CD127, CD8, CD19, CD16 and CD14. The last 4 mAbs were conjugated with the same fluorochrome in aim to cut-off in one step cytotoxic T cells (Tc), B cells, natural killer (NK) cells and monocytes, respectively, that could potentially contaminate isolated CD4⁺ population. These cells were defined all together in sorting algorithm as lineage. Then, cells were sorted with FACS sorter Influx (BD Biosciences, USA) into the following phenotype of Tregs: CD3⁺CD4⁺CD25^HighCD127^−/Lowlin⁻doublet^- and Teffs: CD3⁺CD4⁺CD25^-CD127^Highlin⁻doublet^- as it was reported before^{4 (link)–7 (link),14 (link),60 (link)}.