Joris van Dongen

71 FAT procedure to obtain SVF for regenerative purposes Subset B: CD29-APC (eBiosience, Vienna, Austria), CD44-FITC (BD Bioscience, San Jose, CA, USA) and CD105-Pe/Cy7 (eBiosience, Vienna, Austria). As isotype controls, the following monoclonal antibodies were used: Mouse IgG1 kappa-Pe/Cy7, Mouse IgG1 kappa-APC (both eBioscience, Vienna, Austria) and Mouse IgG1 kappa-FITC (Biolegend, San Diego, CA). Cells were mixed well with each antibody and incubated on ice and in dark for 30 min. Isotype cells were used to set the gates on the basis of the staining. A BD FACScalibur system was used to analyze the samples. Adipogenic, Osteogenic & Smooth Muscle cell differentiation assay Cells from passage 2-4 (n=6) were collected and cultured in a 24-well plate and incubated in DMEM (containing 10% FBS, 1% L-Glutamine and 1% P/S). After reaching confluence, adipogenic, osteogenic and smooth muscle cell differentiation medium was added to show lipid accumulation, mineralization of bone-like noduli and expression of filamentous actin (F-actin) respectively. Adipogenic differentiation medium consisted of DMEM, 0,1µM dexamethasone, 1 nM insulin, 0,5 mM isobutymethylxanthine (IBMX). Osteogenic medium consisted of DMEM, 0.1µM dexamethasone, 10 mM β -glycerophosphate and 0.05 mM ascorbic acid. Smooth muscle cell differentiation medium consisted of DMEM and 0.1% TGF-beta1. Cells were cultured in differentiation medium for 14 days. Medium was refreshed twice a week. After 14 days, cells were fixed with 2% PFA and stained with Oil Red O (Sigma- Alderich, St. Louis, MO, USA) for adipogenic differentiation, Alizarin Red (Sigma- Alderich, St. Louis, MO, USA) for osteogenic differentiation and Phalloidin-FITC (1:250, Invitrogen™, Thermo Fisher Scientific, NY) in DAPI for 30 min for smooth muscle cell differentiation. Oil Red O staining and Alizarin Red staining were evaluated with a light microscope (Leica Microsystems, DM IL). Phalloidin-FITC staining was evaluated with an immunofluorescence microscope (Leica Microcystems, DM IL). Colony formation assay Hundred and thousand cells of FAT-SVF and control (n=3) were plated in a 6-well plate in triplicate and cultured for fourteen days. Cell culture plates were washed with PBS three times and fixed with 2% PFA in PBS for fifteen min. Cells were again washed with PBS three times and stained with 0.05% Crystal Violet (Sigma-Aldrich). After staining, cells were washed with tap water two times and dried inverted. The cloning efficiency or capacity to form colonies from single seeded cells was assessed by determining the area of colonies as well as their intensity of staining with Crystal Violet, both of which are surrogate determinants for the number of cells. Colony area and intensity were analyzed using a plugin for imageJ. 23 Images were taken using Tissue FAXS microscope (TissueGnostics, Vienna, Austria).