Foxp3 fix perm kit

For CD40L intracellular staining, sorted T cells were stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for an hour, fixed (BD CytoFix/CytoPerm™) and permeabilized (BD PERM/Wash™ solution). After permeabilization, cells were stained with anti-CD40L PE (24-31, Biolegend, 1:20 dilution). For IL-10 staining, total lymphocytes were stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for four hours and then for an additional two hours in the presence of Golgi plug (BD). Cells were stained for surface molecules prior to fixation. After fixation, cells were permeabilized and stained with anti-IL-10 Alexa Fluor 647 (JES3-9D7, Biolegend, 1:20 dilution). For BCL6, BLIMP-1, and Ki67 staining, total lymphocytes were stained for surface molecules prior to fixation. After fixation, cells were permeabilized and stained with anti-BCL6 PE (K112-91, BD, 1:50 dilution), anti-BLIMP-1 PE (6D3, BD, 1:50 dilution), or anti-Ki67 FITC (MOCP-21, BD, 1:100 dilution). FOXP3 staining was analogous to that of BCL6 except for changes in fixation and permeabilization (Biolegend FOXP3 FIX/PERM kit) and the use of anti-FOXP3 PE (206D, Biolegend, 1:10 dilution). Samples were loaded on LSR II (BD). Events were collected and analyzed with FlowJo software (TreeStar, CA).

Freshly isolated cells were used for the procedures. Information on antibodies is listed in Key resources Table. Following markers were used to identify the specific population of cells; total leukocytes (CD45⁺), neutrophils (Ly6G⁺), monocytes/macrophages in the gut (CD64⁺Ly6G⁻Ly6C⁺), splenic F4/80^hi macrophage (CD11b⁺F4/80^hiLy6G⁻Ly6C⁺), splenic monocytes (CD11b⁺Ly6G⁻Ly6C^hi), and dendritic cells (DC) (CD11c⁺F4/80⁻CD64⁻). The gating strategy for the SI is shown in Fig. S3A. For intracellular cytokines staining (ICS), cells were treated with phorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Sigma Aldrich) and Ionomycin (1 μg/ml, Sigma Aldrich) for 5-6 hrs with GolgiPlug (BD Biosciences) in the last 4 hrs. Cytofix/Cytoperm kit (BD Biosciences) was used for ICS. FOXP3 Fix/Perm Kit (BioLegend) was used for Foxp3 staining. Apoptotic cells were analyzed using FITC-Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) as recommended by the manufacturer. Cell proliferation was detected by Ki67 staining, where surface-stained cells were fixed and permeabilized using 70% ethanol for 1hr at −20°C followed by staining with Ki67 antibody (16A8, BioLegend). Flow cytometry analysis was performed with BD Fortessa (BD Biosciences), and data were analyzed with FlowJo software (Treestar).

Intracellular cytokine staining was performed on spleen and LN single cell suspensions following 6 hr stimulation with PMA (10ng/ml) (Sigma Aldrich) and ionomycin (1μg/ml) (Sigma Aldrich), with GolgiPlug (BD) in the final 4 hrs of stimulation. Staining was performed using a LIVE/DEAD^™ staining followed by fluorochrome-conjugated Abs for cell surface proteins before fixation and permeabilization (BD Cytofix/Cytoperm Kit). Ab staining for intracellular cytokines was then performed in permeabilization buffer. Intracellular staining for Foxp3 was performed on single cell suspensions from spleen and LNs specifically using a FOXP3 Fix/Perm Kit (BioLegend).

Peripheral blood mononuclear cells (PBMCs) were stained in four panels containing anti-CD3-PacificBlue (PacBlue) (antibodies from BD Biosciences unless otherwise indicated), anti-CD3-Alexa700, anti-CD25-PE-Cy7, anti-CD38-PE, anti-HLA-DR-PE-Cy7, anti-CCR5-APC, anti-CD123-PerCP-Cy5.5, anti-CD16-PacBlue, anti-CD80-FITC, anti-CD83-PE, anti-CD86-APC, anti-PD1-FITC, anti-PD-L1-PE, anti-HLA class I-APC, anti-CD69-APC-Cy7; anti-CD4-Qdot655, anti-CD8-PE-Cy5.5, anti-CD14-Qdot605 (Invitrogen); anti-CD45RA-ECD, anti-CD127-PE, anti-HLA-DR-ECD, anti-CD20-ECD (Beckman Coulter); anti-CD11c-Alexa700 (eBioscience); and anti-CD27-APC-Cy7 (BioLegend). A staining reagent for dead cells (Invitrogen Aqua Live/Dead Fixable Stain) was included. Cells were then washed and fixed in PBS containing 1% paraformaldehyde or permeabilized using a FOXP3 Fix/Perm kit (BioLegend) according to the manufacturer's instructions, intracellularly stained with anti-Ki67-Alexa 488 (BD Biosciences), anti-FOXP3-PacBlue, anti-T-Bet-BV711 (BioLegend), and anti-Eomes-eFluor 660 (eBioscience), fixed, and analyzed with a LSR II cytometer (Becton Dickinson) and FlowJo.