Mouse igg fitc

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).

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).