Cd31 phycoerythrin pe

For flow cytometric analyses, isolated SVF single cells and ad-MVF, which were digested in Accutase® (BioLegend, Fell, Germany) for 30 min into single cells, were used. The single cells were analyzed for the expression of the monoclonal rat anti-mouse endothelial cell marker CD31-phycoerythrin (PE) (BD Biosciences, Heidelberg, Germany), the perivascular cell marker mouse anti-α-SMA (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the monoclonal stromal/stem cell surface markers rat anti-mouse CD117-FITC (BD Biosciences), mouse anti-rat/mouse CD90-FITC (BioLegend) and hamster-anti-mouse CD29-FITC (BioLegend). Isotype identical rat IgG-PE or rat IgG-FITC (BD Biosciences), mouse IgG-FITC (BD Biosciences) and hamster IgG-FITC (BioLegend) served as controls. Additionally, cells were analyzed for the expression of the purified polyclonal sheep anti-mouse/human adipocyte marker ASAM (R&D Systems, Wiesbaden, Germany) followed by a secondary donkey anti-sheep IgG-Alexa488 antibody (Molecular Probes, Eugene, OR, USA). Flow cytometric analyses were performed by means of a FACScan (BD Biosciences) and data were assessed using the software package Cell-Quest Pro (BD Biosciences).

The stromal vascular fraction (SVF) was isolated from aspirated abdominal fat as previously described [14 (link)]. Briefly, each tissue was washed and digested in phosphate-buffered saline containing 0.075% collagenase (Wako Pure Chemicals, Osaka, Japan) for 30 min at 37 °C in a shaking water bath. After centrifugation (800×g for 10 min) and resuspension of the cell pellets, the SVF was obtained by filtering the cell suspension through a series of 100-, 70-, and 40-μm meshes (Millipore, Burlington, MA). Cell counts and viability were measured with an automated cell counter (NucleoCounter NC-100, ChemoMetec, Allerod, Denmark). The SVF samples were examined by flow cytometry using monoclonal antibodies against: CD45-fluorescein isothiocyanate (FITC), CD31-phycoerythrin (PE), CD14-PE, CD34-PE-Cy7 and CD206-allophycocyanin (APC) (BD Biosciences, Franklin Lakes, NJ). Cells were incubated with each antibody for 30 min (dilution, 1:10) and analyzed using a multicolor flow cytometer (MACS-Quant, Miltenyi Biotec) (n = 6 per group). Control gates were set based on staining with a negative isotype control; no more than 0.1% of cells stained positive using the isotype controls.

For flow cytometric analyses, isolated visceral and subcutaneous MVF were further digested into single cells in Accutase® (BioLegend, Fell, Germany) for 20 min at 37 °C. The single cells were then analyzed for the binding of the monoclonal rat anti-mouse endothelial cell marker CD31-phycoerythrin (PE) (BD Biosciences, Heidelberg, Germany), the perivascular cell marker mouse anti-α-smooth muscle actin (SMA) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the monoclonal stromal/stem cell surface markers rat anti-mouse CD117-fluorescein isothiocyanate (FITC) (BD Biosciences), mouse anti-rat/mouse CD90-FITC (BioLegend) and hamster-anti-mouse CD29-FITC (BioLegend). Isotype identical rat IgG-PE or rat IgG-FITC (BD Biosciences), mouse IgG-FITC (BD Biosciences) and hamster IgG-FITC (BioLegend) served as controls. Additionally, cells were analyzed for the binding of the purified polyclonal sheep anti-mouse/human adipocyte marker adipocyte-specific adhesion molecule (ASAM) (R&D Systems, Wiesbaden, Germany) followed by a secondary donkey anti-sheep IgG-Alexa488 antibody (Molecular Probes, Eugene, OR, USA). Flow cytometric analyses were performed by means of a FACScan (BD Biosciences) and data were assessed using the software package CellQuest Pro (BD Biosciences).