Facs aria cell sorter flow cytometer

10–14 week old C57BL/6 males served as gLN donors, and biological triplicates or quadruplicates were collected. Cells were sorted using a FACS Aria cell sorter flow cytometer (Becton Dickinson). MLN dendritic cells were pre-enriched using a Pan Dendritic Cell Isolation Kit (130–100-875, Miltenyi Biotec) and LS MACS Separation Columns (Miltenyi Biotec). Dendritic cells were sorted as Aqua^–CD45⁺Lin^–(CD3^–B220^–NK1.1^–CD19^–) CD11c^hi, and the subpopulations further as MHCII^hiCD103⁺CD11b^– and MHCII^hiCD103⁺CD11b⁺. Stromal cells were not pre-enriched and sorted as Aqua^–CD45^–TER119^–CD24^–TCRβ^–B220^–CD11c^– cells, and the subpopulations further as podoplanin⁺CD31^– (FRCs) and podoplanin⁺CD31⁺(LECs). Three hundred cells were sorted directly into 25 μl TCL buffer (Qiagen, 1031576) supplemented with 1% β-mercaptoethanol at single cell precision. Samples were kept at room temperature for 5 min, spun down and kept at −80 °C until further processing.

Fluorescent-dye-conjugated antibodies were purchased from BD-Pharmingen (USA) (anti-CD45.2, 104; anti-CD45R, RA3–6B2); eBioscience (USA) (anti-CD103, 2E7; anti-MHC II, M5; anti-F4/80, BM8; anti-CD11b, M1/70; anti-CD11c, N418; anti-Siglec F, E50–2440; anti-CD3e,145–2C11; anti-Ly6G, RB6–8C5) or BioLegend (USA) (anti-CD64 X54–5/7.1). Live/Dead staining was performed using Aqua fixable dead cell stain (Invitrogen). Macrophages were sorted as Aqua–CD45+Lin–(CD3–B220–Siglec F–LY6G–) MHCII+F4/80+CD11B+CD11C+CD103–) using a FACS Aria cell sorter flow cytometer (Becton Dickinson).

Naïve (defined as CD4⁺CD25⁻CD62^hiCD44^lo) T cells were sorted using FACS Aria cell sorter flow cytometer (Becton Dickinson) and cultured for 4.5 days in 96 well plates pre-coated with 2μg/ml of anti-CD3ε (17A2) and 1μg/ml of soluble anti-CD28 (37.51). Cells were then stimulated with indicated cytokines (10ng/ml of IL-1β, 20ng/ml of IL-6, 10ng/ml of IL-12, 10ng/ml of IL-23, 10nM of RA, 2ng/ml of TGF-β (Treg), 0.2ng/ml of TGF-β T_H17) in RPMI (Invitrogen) containing 10% FCS (Sigma), 1% L-glutamine (Gibco), 25mM HEPES (Gibco), 1% essential amino acid mixture (Gibco), 5μM β-mercapto ethanol and 1% pen-strep antibiotics (Gibco). Where indicated, cells were stimulated in serum-free media X-VIVO 20 (Lonza) supplemented with the after mentioned components. For in vitro block of leptin signaling, cells were incubated with 250ng/ml of mouse leptin receptor fusioned to Fc portion of immunoglobulin (LepR:Fc chimera (R&D)). For re-stimulation experiments, cells were cultured for 4.5 days as above and resuspended in new media containing the indicated cytokines for another 72h.