Ebioscience foxp3 staining buffer kit

An aliquot (1x10⁶) of total PECs were stained using a viability assay (Zombie UV, Biolegend) and blocked with 5 μg/mL anti-CD16/32 (clone 93; Biolegend) and heat inactivated normal mouse serum (1:10, Sigma-Aldrich) in flow cytometry buffer (0.5% BSA and 2 mM EDTA in Dulbecco’s PBS) before surface staining on ice with antibodies listed in Supplementary Tables 2, 3. For intracellular staining, samples were fixed and permeabilized using eBioscience Foxp3 Staining Buffer kit (Fisher Scientific) following the manufacturer’s instructions before adding antibodies in 1x eBioscience™ Permeabilization Buffer (Thermo Fisher Scientific). All antibodies were purchased from Biolegend unless stated otherwise. PECs were analyzed on a BD FACSymphony machine using BD FACSDiva software (BD Biosciences) and post-acquisition analysis performed using FlowJo v10 software (BD Biosciences). The number of cells per cavity for each population (e.g. F4/80^high resident macrophages) was calculated by determining the percentage of single live cells for each population using FlowJo multiplied by the total number of PECs divided by 100. The gating strategy for flow cytometry analysis is shown in Supplementary Figures 2, 3.

Analysis of IL-10 production was as described previously (23) with a slight modification. Briefly, isolated mesenteric lymph node cells and splenocytes were resuspended (1 × 10⁶ cells/ml) in complete medium (500 ml RPMI 1640 medium plus 10% FCS, 1% L-Glut, 1% Pen/Strep and 5 × 10⁻⁵ M 2-mercaptoethanol (all from Gibco, Carlsbad, CA) with LPS (10 μg/ml, Escherichia coli serotype 0111: B4, Sigma), PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and monensin (2 μM; eBioscience) for 5 h, in 24-well flat-bottom plates. Stained cells were fixed and permeabilised using a Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s instructions and stained with anti-IL-10 (JES5-16E3) or isotype control Rat IgG2b (RT K4530) purchased from Biolegend. Analysis of FoxP3 was performed using eBioscience Foxp3 Staining Buffer kit (Fisher Scientific UK Ltd) following the manufacturer’s instructions.