Foxp3 intranuclear staining buffer kit

The fluorochrome-conjugated antibodies used in this study are listed in Table. S2. Briefly, purified cryopreserved PBMCs were thawed and rested for one hour at 37 °C in complete RPMI medium (RPMI supplemented with Penicillin-Streptomycin, L-Glutamine, HEPES, non-essential amino acids, 2-Mercaptoethanol, and 10% Fetal bovine serum (FBS)). Cells were then washed, resuspended in PBS, and surface stained at 4°C for 20 min with different combinations of antibodies in the presence of fixable live/dead stain (Invitrogen). Cells were then fixed and permeabilized for detection of intracellular antigens. The Foxp3 intranuclear staining buffer kit (eBioscience) was used according to the manufacturer’s instructions for the detection of intranuclear markers. Samples were acquired on a BD Fortessa X20 using BD FACSDiva8.0 (BD Bioscience) and subsequent data analysis was performed using FlowJo 10 (TreeStar). Stochastic neighbor embedding (SNE) analysis was undertaken on the mrc.cytobank platform to enable visualization of high-dimensional data in two-dimensional representations, avoiding the bias that can be introduced by manual gating of specific subsets ^{84 (link)}.

The fluorochrome-conjugated antibodies used in this study are listed in Supplementary Table 2. Briefly, purified cryopreserved PBMCs were thawed and rested for 1 h at 37 °C in complete RPMI medium (RPMI supplemented with penicillin-streptomycin, l-Glutamine, HEPES, non-essential amino acids, 2-Mercaptoethanol, and 10% FBS). Cells were then washed, resuspended in PBS, and surface stained at 4 °C for 20 min with different combinations of antibodies in the presence of fixable live/dead stain (Invitrogen). Cells were then fixed and permeabilized for detection of intracellular antigens. The Foxp3 intranuclear staining buffer kit (eBioscience) was used according to the manufacturer’s instructions for the detection of intranuclear markers. Samples were acquired on a BD Fortessa X20 using BD FACSDiva8.0 (BD Biosciences) and subsequent data analysis was performed using FlowJo 10 (TreeStar). The gating strategies used for flow cytometry experiments are provided in Supplementary Fig. 7a−d. Stochastic neighbor embedding (SNE) analysis was undertaken on the mrc.cytobank platform to enable visualization of high-dimensional data in two-dimensional representations, avoiding the bias that can be introduced by manual gating of specific subsets^{89 (link)}.