Foxp3 af488

Flow cytometry was performed as previously described [38 (link)]. Briefly, cells were incubated with LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue, F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC, CD49b-Pe-Cy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE, FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD), collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).

Peanut-sensitized mice were intragastrically administered twice daily with PBS, sodium butyrate or ButM at an equivalent dose of 0.224 mg g⁻¹ (butyrate per mouse) for 2 weeks. After the treatment, spleen, ileum and colon-draining LNs from mice were collected, and digested in DMEM supplemented with 5% FBS, 2.0 mg ml⁻¹ collagenase D (Sigma Aldrich) and 1.2 ml CaCl_2. Single-cell suspensions were prepared by mechanically disrupting the tissues through a cell strainer (70 μm, Thermo Fisher). Splenocytes (4 × 10⁶) or cells from LNs (1 × 10⁶) were plated in a 96-well plate. Cells were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Thermo Fisher), followed by surface staining with antibodies in PBS with 2% FBS, and intracellular staining according to the manufacturer’s protocols for eBioscience Foxp3/Transcription Factor Staining Buffer set (Invitrogen). The following anti-mouse antibodies were used: CD3 APC/Cy7 (clone 145-2C11, BD Biosciences), CD4 BV605 (clone RM4-5, Biolegend), CD25 PE/Cy7 (clone PC61, Biolegend), Foxp3 AF488 (clone MF23, BD Biosciences), CD11b BV711 (clone M1/70, BD Biosciences), CD11c PE/Cy7 (clone HL3, BD Biosciences), F4/80 APC (clone RM8, Biolegend), I-A/I-E (MHCII) APC/Cy7 (clone M5/114.15.2, Biolegend) and CD86 BV421 (clone GL-1, Biolegend). Stained cells were analysed using an LSR Fortessa flow cytometer (BD Biosciences).