Joris van Dongen

70 Chapter 3 staining of nuclei. Finally, samples were mounted with aqueous mounting agent and visualized under light microscope (Leica Microsystems, DM IL). AMasson’s Trichrome staining was performed as well after 4 µm slides (n=4) were deparaffinized. Samples were mounted with toluene solution and visualized under light microscope (Leica Microsystems, DM IL). Primary antibodies used in this study were directed against alpha smooth muscle actin ( α SMA, 1:200, Abcam, Cambridge, UK) to stain smooth muscle cells, vWF (1:200, DAKO, Glostrup, Denmark) and Perilipin A (1:200, Abcam, Cambridge, UK) to stain adipocytes and. Secondary antibodies used in this study were polyclonal Rabbit anti- Mouse for α –SMA (1:100, DAKO, Glostrup, Denmark), polyclonal Swine anti-Rabbit for vWF (1:100, DAKO, Glostrup, Denmark) and polyclonal Goat anti-Rabbit (1:100, DAKO, Glostrup, Denmark) for Perilipin A. Third antibody used was polyclonal Swine anti-Rabbit (1:100, DAKO, Glostrup, Denmark). Cell Isolation and Culture Ten of the obtained samples (FAT-SVF & control) were washed with PBS three times. After washing, 0.1% collagenase A in PBS/1% bovine serum albumin (BSA) as dissociation medium was added. The samples were stirred for 1.5 hour in a 37°C water bath. Cells were resuspended in lysisbuffer and placed on ice for 5 min to disrupt erythrocytes. The samples were centrifuged at 8°C, 600xg for 10 min and resuspended in DMEM (BioWhittaker Walkersville, MD): 10% Fetal Bovine Serum (FBS), 1% L-Glutamine, 1% Penicillin/Streptomycin (P/S). Then seeded in a 6-well plate or 25cm 2 tissue culture flask depending on the amount of cells. Cells were counted upon staining with Trypan-blue in a Bürker Turk counting chamber. Cells were cultured at 37°C at 5% CO 2 in a humidified incubator, medium was refreshed twice a week. Morphology of the cells derived from the FAT-SVF and control were compared with after 8 days of culture and visualized with an inverted light microscope with Nomarski phase contrast (Leica Microsystems, DM IL). Flow cytometry Cells (n=6) collected from cultures after passage 2-4 were analyzed for CD surface marker expression using flow cytometry. Cells were stained with two different sets of anti-human monoclonal antibodies. Subset A: CD31- phycoerythrine/cyanine7 (Pe/Cy7; IQ Products, Groningen, the Netherlands), CD45-fluorescein isothiocyanate (FITC; IQ products, Groningen, the Netherlands) and CD90-allophycocyanin (APC; BD Bioscience, San Jose, CA, USA).