Cytofix

After tissue disaggregation into a single cell suspension, cells were first stained with a panel of fluorescently labelled antibodies to surface antigens, washed with FACS wash buffer (1% bovine serum albumin (BSA), 0.01% NaN₃ in PBS) then fixed using CytoFix (eBiosciences Foxp3 staining buffer set # 00-5523-00) for a minimum of 30 min at 4 °C. After three washes in perm wash (eBioscience Foxp3 staining buffer), intracellular antigens were stained with another panel of fluorescently labelled antibodies, and cells were then washed with perm wash and analysed by flow cytometry (BD LSR Fortessa X20) and FlowJo software. BD comp beads (anti-mouse Ig k #552843) were used to establish compensation settings. Dead cells were deselected using live/dead stain, added to surface staining panel prior to cell fixation (Life Technologies Live/Dead^TM Near IR fixable dead cell stain kit #L10119). Isotype controls were used during antibody optimisation.

The phenotypes of DCs and BMDMs were determined by flow cytometry. For cell surface staining, single-cell suspensions were incubated for 15 min at 4 °C with PE-anti-CD11c (117308, 0.2 μg/mL), FITC-anti-CD80 (104706, 0.2 μg/mL), PE/Cy7-anti-CD86 (105014, 0.2 μg/mL), FITC-anti-MHC-I (114606, 0.2 μg/mL), PerCP/Cy5.5-anti-MHC-II (107626, 0.2 μg/mL), FITC-anti-F4/80 (123108, 0.2 μg/mL), PerCP/Cy5.5-anti-CD11b (101228, 0.3 μg/mL), PE-anti-CD206 (141706, 0.2 μg/mL), APC-anti-CD115 (135509, 0.4 μg/mL), FITC-anti-PDCA-1 (127007, 0.3 μg/mL), PerCP/Cy5.5-anti-CD44 (103032, 0.2 μg/mL), PE-anti-CD62L (104407, 0.2 μg/mL) (all purchased from Biolegend). Samples were washed and then analyzed by FACS versus flow cytometry (BD Biosciences).
For intracellular staining of cytokines, single-cell suspensions from DLN and spleen of the indicated EAE mice were stained with FITC-anti-CD4 (553047, BD Biosciences, 0.2 μg/mL) first, followed by staining with PE-anti-IFN-γ (554412, BD Biosciences, 0.3 μg/mL), PE-anti-IL17A (506904, Biolegend, 0.3 μg/mL) and PE-anti-IL4 (12-7041-82, eBioScience, 0.4 μg/mL) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol. Intranuclear staining with PE-anti-Foxp3 (126404, Biolegend, 0.4 μg/mL) was performed using a Fixation/Permeabilization kit (eBioscience) according to the manufacturer’s protocol.

Single-cell suspensions were stained with a panel of fluorescently labeled antibodies to surface antigens, washed with fluorescence-activated cell sorting wash buffer (1% BSA, 0.01% NaN₃ in PBS), and then fixed using CytoFix (eBioscience; Foxp3 Staining Buffer Set 00-5523-00) for a minimum of 30 min at 4 °C. We performed three washes in permeabilization wash (eBioscience; Foxp3 Staining Buffer), intracellular antigens were stained with another panel of fluorescently labeled antibodies, and cells were then washed with permeabilization wash and analyzed by flow cytometry (BD; LSRFortessa X-20) and FlowJo software. BD CompBeads (anti-mouse immunoglobulin K; 552843) were used to establish compensation settings. Dead cells were deselected using live/dead stain, added to the surface staining panel prior to cell fixation (Life Technologies; Live/Dead Fixable Near-IR Dead Cell Stain Kit L10119). Isotype controls were used during antibody optimization.