Foxp3 transcription factor fixation buffer

Cells were stained with the antibodies listed in Extended Data Table 1&2. Briefly, after live/dead staining with LIVEDEAD Blue (Thermo Fisher Scientific) on ice for 15 min, cells were washed and incubated with FcR blocker (Miltenyi Biotech) diluted in PBS/2% BSA on ice for 10 minutes. For surface staining, cells were incubated in an antibody mixture diluted in brilliant buffer (BD Biosciences) at 1:100 to 1:250 on ice for 30 minutes. After washing with PBS/2% BSA buffer, cells were fixed with Foxp3/Transcription factor fixation buffer (eBioscience) overnight. For intracellular staining, antibodies were diluted in Foxp3/Transcription factor permeabilization buffer at a ratio of 1:250 to 1:500, and cells were incubated at room temperature for 45 minutes. For intracellular cytokine detection, cells were stimulated using Cell Stimulation Cocktail plus protein transport inhibitor (eBioscience) in complete RPMI for 4 hours, followed by the cell staining procedures described above. Stained cells were acquired with an Aurora (Cytek Biosciences) and analyzed using FlowJo software (v10, BD Biosciences).

For analysis of leukocytes from the immunisation model, spleen or lymph nodes were macerated through 40 μm filters and treated with tris ammonium chloride red blood lysis buffer. Cells were transferred to 96 round bottom plates and washed with PBS.
For analysis of in vitro cultured Th17 cells, cell were transferred to round bottom plates. For intracellular cytokine staining, cells were stimulated with cell activation cocktail (1:500) (Biolegend, 423301), brefeldin A (5 μg/mL) (Biolegend, 420601) and monensin (2 μM) (Biolegend, 420701) for 5 hours prior to staining.
The cells were stained with 30 μL of surface stain prepared in PBS and incubated for 20 minutes on ice. Cells were fixed a using fixation buffer (Biolegend, 420801) for 20 minutes on ice or for nuclear staining, cell were fixed with FoxP3 transcription factor fixation buffer (eBioscience, 00-5523-00) for 1 hour on ice. Prior to intracellular staining, cells were permeabilised using perm/wash buffer (Biolegend, 421002) for 20 minutes on ice and then stained with 20-30 μL of intracellular stain in perm buffer. Samples were analysed on an Attune NxT flow cytometer (Thermofisher Scientific) or a BD Fortessa flow cytometer (BD biosciences).
Gating schemes can be found in Figures S2F and S5E.

For cytokine staining, in vitro-cultured CD4⁺ T cells were stimulated with PMA (Sigma-Aldrich) and ionomycin (EMD Millipore) for 2 h followed by monensin (BioLegend) for a total of 6 h. After fixation with 4% formaldehyde for 10 min at room temperature (RT), cells were washed two times with FACS buffer (PBS with 0.5% BSA). For transcription factor staining, cells were fixed with Foxp3/Transcription factor fixation buffer (eBioscience) at 4 °C in dark for 30 min or overnight. Fixed cells were permeabilized with permeabilization buffer (eBioscience), and stained for cytokines and transcription factors with fluorochrome-conjugated antibodies (1:200 dilution) at 4 °C in dark for 1 h. Stained cells were washed two times with FACS buffer and resuspended with 500 μl of FACS buffer for flow analysis. Fluorescent antibodies for flow cytometric analysis are listed in Supplementary Table 6.