Cd19 1d3

A single cell suspension prepared from colonic tissues as described above was fixed using PBS containing 1% paraformaldehyde and stained on ice for 25 min with antibodies in PBS containing 2% heat-inactivated fetal calf serum. The antibodies used were fluorescein isothiocyanate (FITC)-labeled anti-murine Gr-1 (RB6-8C5, eBioscience), and allophycocyanin-labeled anti-murine CD11b (M1/70, BD Pharmingen). Data collection and analysis were performed on a fluorescence-activated cell sorting (FACS) Calibur flow cytometer using CellQuest software (Becton Dickinson). For cell sorting, a single cell suspension from the colon was stained with antibodies and sorted on a FACS AriaII (Becton Dickinson). The antibodies used for sorting were those described above plus FITC-labeled anti-murine T-cell receptor (TCR)-β (H57-597, eBioscience), Ly-6G (1A8, eBioscience), CD19 (1D3. eBioscience) or CD3 (145-2C11, eBioscience), PE-labeled anti-murine CD49b (DX5, Biolegend), CD11c (N418, BD Pharmingen), or F4/80 (BM8, Biolegend).

After blocking Fc receptors with anti-CD16/CD32 Ab (BD Bioscience), surface staining of isolated single cells was performed in PBS with 2% FBS with the following monoclonal antibodies (mAb) or reagent: PE anti-CD138 (282-2, BioLegend; 1:100 dilution), APC-Cy7 anti-B220 (RA3-6B2, BioLegend; 2:100), 7-AAD (BioLegend; 3:100), FITC anti-GL-7 (GL7, BD Bioscience; 0.5:100), PE anti-PD-1 (J43, BD Bioscience; 1.5:100), APC anti-Fas (15A7, eBioscience), biotinylated mAb for CD8a (53-6.7, BioLegend), CD11c (N418, BioLegend), CD19 (1D3, eBioscience), and CXCR5 (2G8, BD Bioscience; 5:100), and streptavidin (SA)-APC (BioLegend; 1.5:100). For phosphorylated-protein detection, cells were stained with p-Ser235.236-S6 ribosomal protein (D57.2.2E, Cell Signaling; 1:100) and p-Thr37/46-4E-BP1 (236B4, Cell Signaling; 1:100) after preparing with BD Phosflow lyse/fix buffer and perm buffer III (BD Bioscience), according to the manufacturer’s protocol. For MitoTracker staining, cultured cells stained with 30 nM MitoTracker Green (Invitrogen) and 30 nM MitoTracker DeepRed (Invitrogen) were incubated in a CO₂ incubator at 37°C for 30 min, according to the manufacturer’s protocol. Stained cells were analyzed and sorted using a FACSVantage SE (Becton-Dickinson, Franklin Lakes, NJ, USA). Data were further assessed with FlowJo (Tree Star, Ashland, OR, USA).

All antibodies were purchased from Biolegend unless stated otherwise. T cell development were detected by staining with antibodies to CD4(RM4–5), CD8α(53–6.7), CD44(IM7,BD), CD25(PC61), Thy1.2(53–2.1). γδT cell were detected by using anti-γδT(GL3), CD45RG(C363.16A), CD3(145–2C11) and lineage (Lin) CD4(GK1.5), CD8a(53–6.7), NK-1.1(PK136), CD49b(DX5), Ly-6G/Ly-6C(RB6–8C5), CD11b(M1/70), TER-119(TER-119), TCRβ(H57–597), CD19(1D3,eBioscience), B220 (RA3–6B2). T cell activation analysis were done by staining with antibodies to CD4(RM4–5), CD8α(53–6.7), CD25(PC61), Thy1.2(53–2.1). Data were acquired on a BD FacsCanto II flow cytometer in 8-color configuration and cell sorting was conducted using a Beckman Coulter Astrios or MoFlo.

Fc receptors were blocked with anti-CD16/32 (2.4G2, BD Pharmingen). Cell viability was assessed using Zombie Violet dye (Biolegend). Cells were suspended in 1X PBS (pH 7.4) containing 0.01% NaN₃ and 1% fetal bovine serum (i.e., FACS buffer). Surface staining, performed at 4 degrees for 20 minutes, included antibodies specific for murine: Siglec F (E50-2440, BD Pharmingen), CD11b (M1/70), CD64 (X54-5/7.1), CD45 (104), CD3 (17A2, eBiosciences), CD19 (1D3, eBiosciences), CD11c (N418), I-A/I-E (M5/114.15.2), Ly6G (1A8), Ly6C (HK1.4), TNF (MP6-XT22). For ICS, Brefeldin A was added for duration of LPS stimulation. Cyto-Fast Fix/Perm and Cyto-Fast Perm Wash reagents were used for intracellular staining. Reagents are from Biolegend unless otherwise noted. MHC class II tetramers ESAT-6 (I-A(b) 4–17, sequence: QQWNFAGIEAAASA) and MHC class I tetramers TB10.4 (H-2K(b) 4–11, sequence: IMYNYPAM) were obtained from the National Institutes of Health Tetramer Core Facility. Cell sorting was performed on a FACS Aria (BD Biosciences). Sorted cells were collected in complete media, spun down, resuspended in TRIzol, and frozen at -80°C overnight prior to RNA isolation. Samples for flow cytometry were fixed in 2% paraformaldehyde solution in PBS and analyzed using a LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Inc.).

We isolated stromal vascular cells using previously described methods [7 (link)], with some modifications. Mice were sacrificed under general anesthesia after systemic heparinization. eVAT was removed and ground into small pieces. Samples were incubated for 40 min in collagenase II/DNase I solution (1 mg/ml collagenase II and 50 μg/ml in HBSS solution) with gentle stirring. Digested tissue was then centrifuged at 1000 g for 10 min. The resulting pellets were washed twice with cold PBS and filtered through a 70-mm mesh. Red blood cells were lysed with erythrocyte-lysing buffer (Biolegend) for 10 min and resuspended in RPMI-1640 supplemented with 10% FBS. Single-cell suspensions of adipose stromal vascular fraction (SVF) were blocked with CD16/32 monoclonal antibody (2.4G2; BD Biosciences) at 4°C for 5 min. Cells were stained with a mixture of antibodies at 4°C for 20 min. Flow cytometric analysis and sorting were performed on a FACSAriaIII instrument (BD Biosciences) and analyzed using the FlowJo software (Tree Star). Antibodies were specific to CD3 (14A-2; Biolegend), CD4 (GK1.5;Biolegend), CD8a (OKT8; Biolegend), CD44 (IM7; Biolegend), CD11b (M1/70; Biolegend), F4/80 (BM8; Biolegend), PD-1 (29F.1A12, Biolegend), CD19 (1D3, eBioscience), and CD153 (RM153, Biolegend)