Joris van Dongen

284 Chapter 12 DM IL). A Masson’s Trichrome staining and Hematoxylin & Eosin (H&E) staining were performed on deparaffinized four µm of slides. Then, samples were mounted and visualized under a light microscope (Leica Microsystems, DM IL). DNA content measurement NAM and DAM samples (n=3) were used to assess presence of DNA. A solution containing 5 µL proteinase K (2U/mg) (Sigma Aldrich, 3115828001), 50 µL 10% SDS and 500 µL SE-buffer (75 mMNaCl, 25 mMEDTA and pH 8.0) were added to 10 mg of acellular matrix. Solutions were mixed gently and incubated overnight at 55 °C. Then, 6M NaCl and chloroform were added, and samples were thoroughly mixed with the use of a top-over-top rotator for 30 min. Samples were centrifuged at 650 xg at 20 °C for 10 min. Next, supernatant was taken and mixed with ice-cold isopropanol. Samples were centrifuged at 18,000 xg at 4 °C for 5 min. Then, supernatant was discarded, and pellet was washed with 70% ethanol. Afterwards, pellet was dissolved in TE buffer (containing 10 mM Tris and 0.1 mM EDTA, pH 8.0). DNAwas quantified with the use of a Nanodrop meter. Additionally, a DAPI/phosphate buffered saline (PBS) staining was used to stain nuclei. Samples were visualized under a fluorescence microscope (Leica Microsystems, DM IL). Sulphated Glycosaminoglycans measurement The concentration of sulphated glycosaminoglycans (sGAG) was measuredwith the use of a 1,9-dimethylmethylene blue (DMMB) assay according to the protocol of Farndale et al. 24 . NAM and DAM samples (n=3) were digested in 1% proteinase K/SE solution at 55 °C for 24h. As a control, a standard solution of 10 µgl/ml chondroitin sulphate C (#C4384-250 mg, Sigma-Aldrich, St Louis MO) was used. After the addition of the DMMB staining solution, extinction was measured at 525 and 595 nm. Additionally, an Alcian blue staining was used to visualize the total amount of glycosaminoglycans in non-diabetic and diabetic adipose tissue as well as tSVF and NAM and DAM samples. Samples were visualized under a light microscope (Leica Microsystems, DM IL). Cell isolation, characterization and collection of conditioned medium of ASCs Adipose tissue was harvested by normal liposuction procedures from healthy donors (n=3) used for cell isolation. Briefly, samples were washed with PBS three times. Then, 0.1% collagenase A/1% bovine serum albumin (BSA) in PBS was added as dissociation medium. The samples with 0.1% collagenase A/BSA in PBS were then stirred in a water bath at 37 °C for 1.5h. Next, cells were placed in lysis buffer on ice for 5 min. to disrupt all erythrocytes. Cells were then centrifuged and cultured at 37°C at 5% CO 2 in