Clone rb6 8c5

CD4⁺ T cells-depleted splenic cells were cultured with IL-23 (20 ng/ml, Biolegend) with or without butyrate (0.5 mM) for 16 h, and then stimulated with ionomycin (750 ng/ml) and phorbol-12-myristate 13-acetate (50 ng/ml) for 2 h, followed by addition of brefeldin (BD Biosciences) for another 3 h. After Fc blocking, cells were stained with surface markers (PE/Cy7-anti-Thy1, 1:200, Clone#30-H12, Cat#105326; FITC-lineage (CD3, Clone#145-2C11, Cat#100306; CD11b, Clone#M1/70, Cat#101206; CD11c, Clone#N418, Cat#117306; B220, Clone#RA3-6B2, Cat#103206; F4/80, Clone#BM8, Cat#123108; NK1.1, Clone#PK136, Cat#108706; and Gr1, Clone#RB6-8C5, Cat#108419), 1:100), which were purchased from Biolegend. Cells were permeabilized using Foxp3/Transcription Factor Fixation/Permeabilization set (Cat#00-5523-00, ThermoFisher), followed by intracellular staining (anti-IL-22, 1:200, Clone#1H8PWSR, Cat#12-7221-82, Invitrogen; anti-IL-17, 1:200, Clone#TC11-18H10.1, Cat#506916, Biolegend). For ILC3, cells were also stained with RORγt (1:200, Clone#B2D, Cat#17-6981-82, Invitrogen).
All the events were collected by BD FACS Diva software. IL-22 and IL-17 production in ILCs (Thy1⁺ Lineage⁻ cells), or in ILC3 (Thy1⁺ Lineage⁻ RORγt⁺ cells) was analyzed using FlowJo. Gating strategies are included in Supplementary Fig. 15.

Flow cytometry experiments were performed using a BD FACS Fortessa. Populations of mature blood cells were identified by staining PB and BM for B220 (BioLegend, clone RA3-6B2), CD3 (Thermo Scientific, clone 145-2C11), Gr1 (BioLegend, clone RB6-8C5) and Mac1 (BioLegend, clone M1/70). Analysis of erythroid precursors in PB and BM was conducted with antibodies against Ter-119 (BioLegend, clone TER-119) and CD71 (BioLegend, clone R17217). Stem and progenitor cells in BM were identified as previously described [5 –7 ] by staining with a cocktail against lineage markers (BioLegend, B220, CD3, Gr1, Mac1 and Ter119) as well as for c-Kit (eBioscience, clone 2B8), Sca1 (BioLegend, clone D7), CD34 (BioLegend, clone MEC14.7), Fc-γ-II/III-R (eBioscience, clone 93), Thy1.1 (BioLegend, clone OX7) and Flt3 (eBioscience, clone A2F10). Gating strategies were determined by fluorescence minus one staining [8 ].

Spleens were dissociated to a single-cell suspension and then filtered using a 70 µm nylon mesh. Cells were counted using trypan blue exclusion assay. A number of 1 × 10⁶ cells were plated and stained with anti-F4/80 (1:100, cloneBM8; BioLegend, San Diego, CA, USA), CD169 (1:100, clone 3D6.112; BioLegend), CD115 (1:100, clone 3D6.112; BioLegend), Gr1 (1:500, clone RB6-8C5; BioLegend), CD19 (1:100, clone CD5, BioLegend), CD4 (1:100, BM4-5, BD Horizon, BD Biosciences, San Jose, CA, USA), CD8 (1:200, clone 53-6.7, BioLegend), and NK1.1 (1:100, clone Pk136 BioLegend) for 30 min, at 4 °C. Then, cells were washed and fixed with 2% PFA for 10 min at room temperature (RT). Cells were washed and analyzed using a BD FACS Canto II flow cytometer and FlowJo (BD Biosciences).