Cd86 gl1

Single cell suspensions from spleen, liver and visceral adipose tissue were incubated with 2.4G2 mAb (anti-FcγRIII/II) to block non-specific binding of primary mAb and then cells were stained with a combination of the following mAb: FITC conjugated anti-TCRβ (H57-597, BioLegend), anti-CD11b (Mac-1, TONBO Biosciences), anti-CD206 (C068C2, BioLegend), APC conjugated anti-NK1.1 (PK136, BioLegend), anti-CD11c (HL3, BD Pharmingen), PE conjugated anti-α-GalCer:CD1d complex antibody (L363, BioLegend), α-GalCer (PBS-57)-loaded CD1d tetramer kindly provided by NIH Tetramer Core Facility at Emory University (Atlanta) and Brilliant Violet 421 conjugated anti-F4/80 (BM8.1, TONBO Biosciences). 3T3-L1 adipocytes were stripped off by PBS containing 1.5 mM EDTA and stained with PE conjugated anti-CD1d (1B1, BioLegend), anti-CD80 (16-10A1, BD Pharmingen) and -CD86 (GL1, BD Pharmingen). Stained cells were sorted using FACS Aria (BD Biosciences) or assessed using FACS Caliber flow cytometers (BD Biosciences). Data were analyzed with FlowJo software (FlowJo, LLC).

Lymph nodes were processed into single cell suspensions using a 70 μm cell strainer. Corneas were excised and incubated with 1 mg/ml collagenase D (11088866001, Roche, Switzerland) for 1 h at 37°C to obtain single cell suspensions. Cells were incubated with Fc block (CD16/CD32) for 15 min at 4°C, followed by incubation with directly conjugated monoclonal antibodies (mAb) to CD3 (145-2C11), CD4 (GK1.5), CD45 (30-F11), CD11b (M1/70), CD11c (HL3), Gr-1 (RB6-8C5), CD40 (3/23) and CD86 (GL1) (all from BD Biosciences, USA) for 30 min at 4°C. For staining of intracellular transcription factor, cells were incubated with Cytofix/Cytoperm (BD Biosciences, USA) for 20 min at 4°C followed by staining with directly conjugated, T-bet (4B10, eBioscience, UK) and RORγt (Q31-378, BD Biosciences, USA) for 30 min at 4°C. Thereafter, the cells were washed and data acquired with a BD LSRII Flow Cytometer (BD Biosciences, USA). Data analysis was performed using the FlowJo software (Miltenyi Biotec, Germany). For calculation of absolute cell numbers, the percentage cell population was multiplied by total cell count.

Fluorescence-activated cell sorting (FACS) analysis was carried out as described previously30 (link). Briefly, cells in the SVF were suspended in FACS buffer and incubated with anti-mouse CD16/CD32 (93; Biolegend, San Diego, CA) for 15 min. Then, the cells were rinsed and resuspended in FACS buffer and stained with anti-CD11c (HL3; Biolegend) and anti-CD11b (M1/70: Biolegend) antibodies for DC and macrophages, respectively. The expression of co-stimulatory molecules was determined using mAbs to CD80 (16-10A1; BD), CD86 (GL1; BD), CD40 (3/23; BD), and MHC class II (M5/114.15.2; BD). The expression of other surface antigens was analyzed by using mAbs to F4/80 (BM8; Biolegend), CD103 (M290; BD), CD205 (NLDC145; MACS), CD206 (MR5D3; AbD Serotec), mPDCA1 (JF05-1C2.4.1:MACS), and B220 (RA3-6B2; Biolegend). To detect lipid, cells were first stained with 1 μg/ml Nile red (Wako Pure Chemicals, Osaka, Japan) for 15 min, and then analyzed with FACSVerse (BD Biosciences). The expressions of Treg related molecules were determined by using mAbs to CD25 (PC61; Biolegend), FR4 (12A5; Biolegend), CTLA4 (UC10-4B9; Biolegend) and GITR (DTA-1; Biolegend).