Nkg2d pe

The cultures were collected, washed, incubated for 15 min with mouse mAbs against human CD3-PerCP, CD56-FITC, or PE, CD69-APC, CD16-PE (BD Biosciences, USA), and NKG2D-PE (BioLegend, USA). NK cells were incubated with CD158a-PE and CD158b-PE (BD Pharmingen, USA), CIK cells were incubated with CD4-PE and CD8-APC (BD Biosciences) and γδ T cells were incubated with Vγ9-FITC (BD Pharmingen), CD4-PE, and CD8-APC. Isotype-matched antibodies were used as controls. Perforin and granzyme B detection was performed according to the BD Cytofix/Cytoperm™ Kit manual (BD Biosciences). Briefly, NK, CIK, and γδ T cells were harvested and adjusted to 1 × 10⁶ cells/mL in RPMI-1640 medium containing 10 % fetal calf serum, and incubated 0.1 % GolgiStop (BD Biosciences) for 4 h. After pre-incubation with 10 % normal human serum, cells were stained with mAbs to identify NK (CD3⁻CD56⁺), CIK (CD3⁺CD56⁺), and γδ T cells (CD3⁺Vγ9⁺), followed by intracellular staining for perforin-PE and granzyme B-PE (BD Pharmingen), and the corresponding isotype antibodies to determine intracellular cytokine levels.
Flow cytometry data acquisition was performed on a BD FACS Calibur (BD Biosciences) with Cell Quest Pro software. Analysis was performed with FlowJo software (Tree Star, USA).

Single-cell suspensions from the SVF of fat tissue, spleen, and blood samples were incubated in FcBlock (BD Biosciences) for 15 min at 4°C. The cells were then stained with fluorophore-conjugated antibodies for 20 min at 4°C in the dark. The antibodies were purchased from BD Biosciences (CD8-FITC, CD11c-FITC, NK1.1-FITC, CD4-PE/Cy7, CD45.2-APC), eBioscience (San Diego, CA) (NKG2D-PE, F4/80-PE/Cy7, CD11b-APC/eFluor780), or BioLegend (San Diego, CA) (CD3ε-Pacific Blue). Dead cells were excluded with 7-AAD staining (BD Biosciences). Flow cytometry data was acquired with FACSCantoII or LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo (Treestar, Ashland, OR).

Staining was performed to determine cell viability (Flexible Viability Dye eFluor™ 780; ThermoFisher) and expression of extracellular/intracellular markers using the following fluorescently-labelled antibodies against human proteins: CD4 (FITC Mab OKT4; ThermoFisher), CD8 (BV711/clone SK1, BioLegend), CD40L (BV605; BioLegend), CD86 (PE/Cy7; BioLegend), NKG2D (PE; BioLegend), CD25 (PE/Cy7; BioLegend, CD127 (eFluor450/eBioRDR5; ThermoFisher) within different cell populations. For intracellular staining Brefeldin (3 µg/mL)(Life Technologies) was added to cultures four hours prior to flow cytometry and intracellular staining for Ki67 (Alexa Fluor 488; BioLegend) within CD4+/CD8+ T cells and iMoDC populations and staining for intracellular IFN-γ (PE clone B27; BioLegend). Staining for T regulatory (Treg) cells was performed using anti-Foxp3 (PE conjugate; BioLegend) within CD4+/CD127^low/CD25+ T cells following fixation and permeabilization (Foxp3 transcription factor fixation buffer; ThermoFisher).