Anti foxp3 pe

Intracellular staining was performed to assess expression of FoxP3, as well as markers associated with the activated state of Treg cells (anti-Helios, anti-neuropilin-1, a transmembrane glycoprotein, and anti-Areg). To assess production of proinflammatory and anti-inflammatory mediators; and the proliferative response of microglia, intracellular staining was performed. We evaluated the expression of interleukin (IL)-6, IL-1β, iNOS (inducible nitric oxide synthase), Arg1 (Arginase1), Areg, and Ki67. BMNC (2 x 10⁶ cells/well) were surface stained prior to fixation/permeabilization using cytofix/cytosperm kit (eBioscience now Thermo Fisher Scientific). Cells were then stained using anti-FoxP3-PE, anti-IL-6-PerCP-eFluor450, anti-iNOS-PE, anti-Helios-eF450, and anti-Ki67-PECy7 (Thermo Fisher Scientific), anti-IL-1β- BV711 (Biolegend, San Diego, CA), Arg1-FITC, anti-Areg-biotinylated-streptavidin-APC (R&D Systems, Minneapolis, MN) and anti-neuropilin-BV650 (BD Bioscience) as recommended by the manufacturer’s protocols. Stained and fixed cells were analyzed using flow cytometry as described above.

Splenocytes were isolated from the spleens of 15–20-week-old C57BL/6 and Roquin^san/san mice. B- and T-cell populations were identified using specific antibodies (eBioscience; San Diego, CA, USA). The 5 × 10⁵ T cells or B cells were stimulated for 4 h ex vivo with PMA (25 ng/ml, Sigma-Aldrich, St Louis, MO) and ionomycin (250 ng/ml, Sigma-Aldrich) in the presence of GolgiStop (BD Biosciences, Sparks, MD). To examine B-cell populations, splenocytes were stained with anti-B220- allophycocyanin (APC), anti-CD21-fluorescein isothiocyanate (FITC), anti-CD23- phycoerythrin (PE), anti-CD138-PE, anti-IgD-FITC, anti-IgM-PE, anti-CD1D-PE, anti- CD19-peridinin chlorophyll protein complex (PerCP), and anti-IL-17-PE antibodies. To analyze T helper and regulatory T (Treg) populations, splenocytes were stained with anti-CD4-PerCP, anti-IL-17-PE, anti-IL-4-PE, anti-interferon (IFN)-γ-FITC, anti-CD25-APC, and anti-Foxp3-PE antibodies. To analyze T follicular helper (TFH) cell populations, splenocytes were stained with anti-CD4-eFluor450, anti-CXCR5-PerCP-eFluor 710, anti-PD1-FTIC, anti-ICOS-PE cyanine7, anti-BCL6-APC, anti-IFN-γ-PE, anti-IL-4-PE, anti-IL-17-PE and anti-Foxp3-PE antibodies (Thermo Fisher Scientific, Waltham, MA). Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, IN, USA). The expressions of cell population were analyzed by CytExpert 2.3 software.

Bone marrow cells from one femur and mesenteric lymph nodes cells were isolated and erythrocytes in bone marrow were lysed using 0.83% ammonium chloride. Cells were stained with eBioscience Fixable Viability Dye eFluor 780 according to the manufacturer’s protocol (Invitrogen, ThermoFisher Scientific). Cells were extracellularly stained with anti-CD3-BV510 (Clone 17A2, Nordic BioSite AB, Täby, Sweden), anti-CD4-FITC (Clone RM4-5, Nordic BioSite AB, Täby, Sweden), and anti-CD25-APC (Clone 3C7, BD, Franklin Lakes, NJ). Cells were fixed and permeabilized using the FoxP3 staining buffer kit (Invitrogen, Thermofisher Scientific) and intracellularly stained with anti-Foxp3-PE (Clone FJK-16s, ThermoFisher Scientific) according to manufacturer’s instruction. Treg cells were defined as CD4+CD25+Foxp3+, and results are expressed as frequency of live cells or number of cells. Samples were run on BD FACSVerse (BD, Franklin Lakes, NJ), and data were analyzed using FlowJo software (version 10.4.1).

Before cytokine staining, T cells underwent in vitro stimulation with PMA (40 ng/ml), Ionomycin (2 μM), Monensin (4 μM) in 1 ml RPMI 1640 (containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 0.1 mg/ml streptomycin, and 10 mM HEPES) for 4 h. Cells were resuspended in PBS supplemented with 0.5% BSA and 1 mM EDTA (FACS buffer) (splenocytes and lymphoid cells: 1 × 10⁶/tube, all available CNS mononuclear cells from each sample/tube) and stained with the antibodies listed below at 4˚C for 30 min. The anti-mouse antibodies used for flow cytometric analysis were the following: anti–CD45–APC-Cy7, anti-CD4-APC, anti-IFNγ-BV421, anti-IL-17–PerCP Cy5.5, anti-IL-10–FITC, anti-Foxp3-PE, anti-CD11b-PE-Cy7, anti-Ly6G-BV421, anti-MHC II-FITC, anti-Ly6C-PerCP Cy5.5, anti-CCR2- BV510 (all antibodies were from eBiosciences or BioLegend). After staining, the cells were analyzed with FACSVerse flow cytometer. Flow cytometric data were viewed and analyzed by FlowJo v10.

Flow cytometric analysis was performed using an LSR II flow cytometer (BD). Cell phenotypes were analyzed using combinations of anti-GITR-PE or -efluor450, anti-PD-1-PE, anti-FoxP3-PE, -efluor450 or -APC, anti-CD8-PE, anti-C45.1 PECy7, anti-Helios-FITC, anti-Eos-PE, anti-CD103-APC, anti-CD4-AlexaFluor700 (all from eBioscience), and anti-Neuropilin-1-PE or -APC, anti-CD103-PerCPCy5.5 (Biolegend) antibodies. Fixable viability dye efluor780 (eBioscience) was used in all experiments to exclude dead cells. Cell proliferation dye (CPD)-efluor450 and CFSE (both from eBioscience) were used to monitor cell proliferation. Anti-Ki67-PE (eBioscience) was used to identify dividing cells. Results were analyzed using FlowJo analysis software (Tree Star, Inc.).

Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Whole blood was drawn in EDTA tubes and the cells were incubated with fluorochrome-conjugated antibodies against human CD3-FITC, HLA-DR-FITC, CD45RA-FITC, CD8-PE, CD16+56-PE, CD69-PE, CD28-PE, CD3-PerCP, CD45-PerCP, CD45RO-PerCP-Cy5.5, CD4-PE-Cy7, CD25-PE-Cy7, CD25-APC, CD39-APC, CD62L-APC, CD8-APC-H7, CD4-APC-H7, CD3-Horizon V450, CD56-Horizon V450, all from BD Biosciences (San Jose, USA). Anti-Foxp3-PE was purchased from eBioscience (San Diego, USA) and Helios-Pacific Blue from Biolegend (San Diego, USA). Lymphocyte subpopulations from peripheral blood were measured by 4- or 7-color combinations. TruCount^™ tubes (BD Biosciences) were used for assessment of absolute numbers (cells/μl) of leukocytes (CD45), and major lymphocyte populations; T lymphocyte (CD3), T helper (CD4), T cytotoxic (CD8), B (CD19) and NK (CD16+CD56) cells, as described by the manufacturer. Absolute numbers of subpopulations were derived from the major populations. Proportions of major populations were given as % of lymphocytes and proportions of subpopulations were given as % of their parent population.