Anti foxp3 antibody

Intracellular cytokine staining and flow cytometry were modified from our previous report [30 (link)]. Briefly, on day 42, single cell suspensions from inguinal lymph nodes (ILNs) were pulsed with 20 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA) and 1 μg/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 18 h, with 10 μg/mL brefeldin A was added during the last 4 h of culture. After stimulation, the cells were surface stained with phycoerythrin- (PE-) anti-CD4 antibody (BD Biosciences, San Diego, CA, USA), permeabilized/fixed with cytofix/Cytoperm Plus (BD Biosciences), and stained with FITC-anti-IL-17A antibody and FITC-anti-IFN-γ antibody (Biolegend). To analyze regulatory T cells (Tregs), single cell suspensions from ILNs were stained with PE-anti-CD4 antibody (BD Biosciences), fixed, permeabilized, and stained with anti-Foxp3 antibody (BD Biosciences) according to the manufacturer’s instructions. Flow cytometer analysis was performed in an AccuriTM C5 cytometer.

A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).

For Treg cells, PBMCs were first stained with anti-CD3 antibody (BioLegend), then fixed and permeabilized. Cells were then stained with anti-FOXP3 antibody (BD Biosciences) for 1 hour before washing and acquisition. The T lymphocyte subpopulations were detected with the antibodies against CD3 (BioLegend), CD4 (BioLegend), CD8 (BioLegend), CD45RA (BD Biosciences) and CD27 (BD Biosciences). Cells acquisition was performed using a FACSCanto II flow cytometer, and the data were analyzed using FlowJo software.