Anti tim 1 pe

CB was from Merck-Millipore, Darmstadt, Germany. Anti-CD3 mAb, anti-CD28 mAb, anti-phosphotyrosine (anti-pTyr) PY20-Alexa 488, anti-CD4-Alexa 488, anti-CD25-PercP, anti-FOXP3-Alexa 488, and anti-TIM-1-PE were from Biolegend, San Diego, CA, United States. Rabbit anti-mouse-IgG1 and IgG2a were from Fisher Biotec, Hampton, New Hampshire, United States. Carboxyfluorescein diacetate succimidylester (CFSE) was from Biolegend. The 7-Plex T-Cell Receptor Th17 and IL-17 Magnetic beads MAGPIX Kits were from Merck-Millipore. Treg (CD4+CD25+CD127^low) and CD4+ T cell isolation kit were from Miltenyi Biotec, Bergisch Gladbach, Germany. The primers were from IDT, Coralville, Iowa, United States. The DNA Extraction and Purification Kit and Gel and PCR Clean-up System were from Promega, Madison, Wisconsin, United States. Recombinant Taq DNA Polymerase was from Thermo Fisher Scientific, Waltham, Massachusetts, United States, and QIA Quick PCR purification kit was from Qiagen, Hilden, Germany.

After 4 h of stimulation, PBMCs were stained with a combination of the following antibodies from BD Bioscience: anti-CD19-PerCP (cat #345–778), anti-CD43-FITC (cat #555–475), anti-CD27-PECy7 (cat #560–609), anti-CD24-FITC (cat #555–427), anti-CD38-PECy7 (cat #335–825), anti-CD25 FITC (cat #555–431), anti-IL-10 APC (cat #554–707) or anti-IL-10-PE (cat #559–330). Anti-CD5 APC was from Dako (cat #C7242, Glostrup, Denmark) and anti-TIM-1 PE was purchased from Biolegend (cat #353–904).
After 48 h of stimulation, IL-10 was detected with the following BD Bioscience Abs: anti-CD19 APC (cat #555–415), anti-CD14 FITC (cat #555–397), anti-CD4 PerCP (cat #345–770), anti-CD8 PECy7 (cat #557–746) and anti-IL-10 PE (cat #559–330). Live/Dead Fixable Near InfraRed staining (cat #L10119; Molecular Probes, Invitrogen, Carlsbad, CA) was included.
The cells were acquired with a FACS Canto (BD Bioscience) flow cytometer with argon laser (488 nm) and Helium-Neon laser (633nm) excitation.
All analyses were carried out using FlowJo V10 (TreeStar, Ashland, OR). Dead cells were excluded based on Live/Dead Fixable Near InfraRed staining and B cells were identified as CD19⁺ events within a morphological lymphocyte gate. Individual IL-10⁺ B cells were identified using the gating strategy demonstrated in S1 Fig.