Anti cd206 c068c2

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).

Tissues were excised from mice, minced and digested in Collagenase for 30 min. The samples were filtered through a 40-μm filter. Erythrocytes were removed with Lysing buffer (BD Biosciences). The samples were then incubated for 10 min with anti-CD16/CD32 blocking antibodies (BD Biosciences). The cells were stained with the following antibodies: anti-CD206 (C068C2, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD11b (M1/70, BioLegend), anti-CD45 (30-F11, eBioscience), anti-F4/80 (CI: A3-1, BD Bioscience), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD4 (GK1.5, BioLegend), anti-TCRβ (H57-597, BioLegend), anti-PD-1 (29F.1A12, BioLegend), and anti-CD44 (IM7, BioLegend). The samples were washed, incubated with 7-amino-actinomycin D (BD Biosciences) and then analysed on a FACSAria II (BD Biosciences).

Antibodies for iNOS (D6B6S) and β-Actin (8H10D10), used for immunoblotting assays, were from Cell Signaling Technology (Danvers, MA, USA). Antibodies used for flow cytometry assays are fluorescently labeled. Fluorescently labeled antibodies for mouse F4/80 (BM8), mCD11b (M1/70), mCD8 (53–6.7), mCD86 (GL1), mCD80 (16-10A1) were purchased from eBioscience (San Diego, CA, USA). Fluorescently labeled antibodies for mCD45 (TU116), mCD44 (IM7), CD62L (MEL-14) were ordered from BD Biosciences (San Diego, CA, USA). Fluorescently labeled anti-mCD4 (RM4-4) was ordered from Biolegend (San Diego, CA, USA).
Cytofix/Cytoperm Golgi Stop Kit with BS GolgiStop™ used for intracellular staining was purchased from BD Biosciences (San Diego, CA, USA). Fixation/Permeabilization Solution Kit used for Foxp3 staining was from eBioscience (San Diego, CA, USA). Fluorescently labeled anti-TNFα (MP6-XT22), anti-IFNγ (XMG1.2) anti-IL-12p40 (C8.6), Foxp3 (NRRF-30) was purchased from eBioscience (San Diego, CA, USA). Anti-CD206 (C068C2) was from Biolegend (San Diego, CA, USA). Fluorescently labeled antibodies to Glut1 (EPR3915). Primary anti-succinate dehydrogenase subunit A (SDHA; 2E3GC12FB2AE2; used for flow cytometry analysis) and goat anti-rabbit IgG H&L (Alexa Fluor® 488) was purchased from Abcam (Mountain View, CA, USA).