Joris van Dongen

129 Isolation of SVF by the FAT procedure Fractionation of adipose tissue procedure 1. 10 ml Luer Lock syringe. 2. Centrifuge with the capability to go to 956 xg (Medilite, Thermo Fisher Scientific, Waltham, MA). 3. Gauge. 4. Fractionator (Luer to Luer connector with three holes of 1.4mm inside) (Tulip, Medical Products, San Diego, CA). Live/dead assay of tSVF 1. 0.001% carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, ThermoFisher #V12883,Waltham,MA) /serum free Dulbecco’s modified eagle’s medium (DMEM, Breda, The Netherlands). 2. 0.001% propiumiodide (PI, ThermoFisher #P3566, Waltham, MA)/serum free DMEM. 3. 2% PFA. 4. 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies #D1306, Carlsbad, CA). Histological characterization of tSVF 1. 10% formalin. 2. 1.8% agarose solution (Select agar, Invitrogen, Life Technologies Corp., Carlsbad, CA, USA). 3. Xylol. 4. 100%, 96%, 70% alcohol. 5. 0.1 MTris/hydrochloric acid (HCl) buffer pH 9.0. 6. 10 mMTris/ 1 mM ethylenediaminetetraacetic acid (EDTA) buffer pH 9.0. 7. 3% hydrogen peroxidase/phosphate buffered saline (PBS). 8. α -smooth muscle actin (SMA, Abcam #ab7871, Cambridge, United Kingdom). 9. Perilipin A (Abcam #ab3526, Cambridge, United Kingdom). 10. Von Willebrandfactor (vWF, Dako #A0082, Glostrup, Denmark). 11. 1% Bovine serum albumin (BSA). 12. 1% Human serum.