Cd4 cd25 cd127lo regulatory t cell isolation kit 2

Single cell suspensions of human FL tumors were cultured with IL-2 (300 IU/ml) in RPMI medium supplemented with 10% human serum plus 1% penicillin-streptomycin. CD8⁺ T cells were depleted by CD8 micro beads (Miltenyi Biotec) and remaining cells were stained and sorted on a FACSAria into two fractions of CD4⁺CD127^loCD25^hi and CD4⁺CD127⁺CD25^lo (Teffs). Tumor cells used as APCs were obtained from suspensions of FL tumors by depleting T cells with CD3 micro beads (Miltenyi Biotec). In selected experiments Tregs were isolated with the CD4⁺CD25⁺CD127^lo/– Regulatory T Cell Isolation Kit II (Miltenyi Biotec), CD4⁺CD25^- Teffs were purified by depleting CD25⁺ cells from CD4⁺ T cells after isolating CD4⁺ T cells using CD4⁺ T-cell isolation kit (Miltenyi Biotec), and CD4⁺CD45RA⁺ naïve T cells were isolated by Naive CD4⁺ T cell Isolation Kit II (Miltenyi Biotec).

Treg cells were isolated from PBMCs using the CD4⁺CD25⁺CD127^lo/− regulatory T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Briefly, non-CD4⁺ cells and CD127^high cells were first depleted using a cocktail of biotinylated antibodies and anti-biotin microbeads. CD4⁺CD25⁺CD127^lo/− cells were directly labeled with CD25 microbeads and isolated over a selection column. The purity of isolated Treg cells and CD25-depleted PBMCs was examined by flow cytometry after staining with fluorochrome-conjugated antibodies to CD3, CD4, CD25, CD127, and Foxp3.