Human il 2 enhanced sensitivity flex set

Pooled cells from LNs and spleens were depleted of B cells and accessary cells by panning and T cells were enriched. The cells were stained with anti-CD8 and anti-B220 Abs, and CD4⁺ T_reg cells were sorted on the basis of GFP (YFP) expression alone in CD8-negative population. The sorted cells were divided among 8 wells of a 96-well V-bottomed plate (2 × 10⁵ cells/well) in 25 μl RPMI medium (10% FCS) with or without increasing doses of recombinant human IL-2 (0.016 to 12 U/ml), followed by incubation for 2 h at 37 °C. Depletion of IL-2 from the medium was assessed with the BD Cytometric Bead Array and Human IL-2 Enhanced Sensitivity Flex Set according to the manufacturer's instructions (BD Biosciences).

Foxp3^CreERT2Cα^FL/WT mice were treated with tamoxifen on days 0 and 1, and on day 9 pooled spleens and lymph nodes were enriched for CD4⁺ cells and subsequently sorted to >99% purity into eGFP⁺TCRβ⁺, eGFP⁺TCRβ⁻, and eGFP⁻TCRβ⁺ populations. Each population was divided among 8 wells of a V-bottom 96-well plate (250,000 cells/well) in 25uL RPMI(10%) with or without increasing doses of recombinant human IL-2 for 2 hours at 37°C. IL-2 depletion from the media was assessed using the BD Cytometric Bead Array and Human IL-2 Enhanced Sensitivity Flex Set (BD Biosciences) according to the manufacturer’s instructions.