Cd25 apc bc96

To investigate the role of T regulatory cells in natural tolerance to foods, cell surface and intracellular cytokine staining was performed using validated methods [23 (link)]. Unstimulated and PBMCs stimulated with ovalbumin and peanut extract were surface stained with markers from the indicated sources (CD3 APC-CY7 (SK7), Biolegend; CD4 V-500 (RPA-T4), BD; CD14 Alexa Fluor-700 (HCD14), Biolegend; CD19 PE (HIB19), BD; RORgT PE (AFKJS-9), eBiosciences; CD25 APC (BC96), Biolegend; and CD127 Pacific Blue (A019D5), Biolegend) and then washed, fixed, and permeabilized for intracellular staining (Foxp3 Alexa Fluor 488 (206D), Biolegend; IL-10 PE-CY7 (JES3-9D7), Biolegend; IL-5 APC (TRFK5), Biolegend) according to manufacturer’s instructions (Caltag, Carlsbad, CA). Acquisition was performed using an LSR-II (BD, San Jose, CA), and analyzed using FlowJo software (Treestar, San Carlos, CA). Data are expressed as the mean percentage of cells within the gated CD3CD4 T cells unless otherwise indicated.

On day −1, NK cells and Tregs from the same donor were sorted as described above in section 2.7. Tregs were rested in cRPMI overnight without stimulation or cytokine treatment. NK cells were activated with IL-12 (1 ng/mL) and IL-18 (1 ng/mL) overnight. On day 0, Tregs and activated NK cells were washed and co-cultured in cRPMI at 1:0, 1:1, 1:10 and 0:1 Treg to NK ratios with 50 U/mL of IL-2 at 37°C for 15 minutes. Cells were fixed with 3% formaldehyde (Polysciences, Inc.) for 5 minutes at 37°C followed by 5 minutes on ice. Cells were then transferred to FACS tubes, washed, and permeabilized by resuspending in 500 μL ice-cold 90% methanol. Cells were stained with antibodies against pSTAT5-PE (pY694, BD Biosciences), CD56-BV605 (HCD56; BioLegend), CD4-Pacific Blue (RPA-T4; BioLegend), and CD25-APC (BC96; BioLegend) for 30 minutes on ice. Samples were collected on a BD LSRFortessa and analyzed using FlowJo software.