Anti il 10 fitc clone jes5 16e3

Leukocytes from SGs and spleens were stimulated with peptides at a final concentration of 3 μg/ml for 2 hr at 37°C. The following peptides were used to stimulate CD4⁺ T cells: m09 residues 133–147 (GYLYIYPSAGNSFDL), M25 residues 409–423 (NHLYETPISATAMVI), m139 residues 560–574 (TRPYRYPRVCDASLS), and m142 residues 24–38 (RSRYLTAAAVTAVLQ). The cultures were then supplemented with brefeldin A (2 μg/ml, Sigma-Aldrich) and incubated for a further 4 hr at 37°C. After stimulation, cells were labeled using a Zombie Aqua Fixable Viability Kit (BioLegend) and stained with anti-CD16/CD32 (Fc block, BioLegend) followed by anti-CD4–APC-Cy7 or anti-CD4–BV605 (clone RM4-5, BioLegend). Internal antigen expression was determined after fixation/permeabilization in BD Cytofix/Cytoperm Solution (BD Biosciences). Cells were stained with combinations of anti-IFNγ–APC-Cy7, anti-IFNγ–FITC, or anti-IFNγ–Pacific Blue (clone XMG1.2, BioLegend), anti-IL-10–APC or anti-IL-10–FITC (clone JES5-16E3, eBioscience), and anti-T-bet–BV605 (clone 4B10, BioLegend). Cells were acquired using an Attune NxT (Thermo Fisher Scientific), and data were analyzed using FlowJo v10.5.3 (FlowJo LLC).

After culture, cells were collected in PBS containing 2% FBS and 1mM EDTA. Live cells were determined with LIVE/DEAD^™ Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Fc receptors were blocked using anti-CD16/CD32 (clone 93, eBioscience) and samples were stained with anti-CD4-Alexa Fluor 700 (clone GK1.5), anti-Foxp3-PE (clone 150D/E4), anti-RORγt-APC (clone B2D), anti-IL17A-PerCP/Cy5.5 (clone eBio17B7), and anti-IL10-FITC (clone JES5-16E3) (all from eBioscience). Samples were fixed and permeabilized by using Foxp3 Transcription Factor Staining Buffer (eBiosience). At least 10⁵ cells were acquired with a Gallios flow cytometer and analyses were performed with Kaluza Analysis (version 1.3) (Beckman Coulter, Krefeld, Germany) and FlowJo softwares (version 10.6.2) (Ashland, OR, USA) following the gating strategy shown in Supplementary Figure 1.