Collagenase nb6

A 6-mm RDEB skin biopsy was obtained with authorization from the National Research Ethics Services, Westminster (07/H0802/104), and with written informed consent from patients with RDEB-1 ((+/–) c.1732C>T p.R578X)/(+/–) c.2710+2T>C IVS20+2T.C) and RDEB-2 ((+/+) c.425A>G p.K142R). Excess connective tissue was removed using a sterile blade and the sample was incubated in neutral protease NB (1 unit/ml; SERVA Electrophoresis, Heidelberg, Germany) at 37 °C for 3 hours until the epidermis peeled off. The remaining dermis was fragmented and treated with collagenase NB6 (0.45 units/ml; SERVA Electrophoresis). The resulting cell suspension was seeded into a T25 flask and cultured at 37 °C in a 5% CO₂ incubator.

Small portions of the intra-abdominal and subcutaneous adipose tissue from mice were digested with 0.225 U/mL collagenase NB 6 (#17458, SERVA Electrophoresis; Heidelberg, Germany) for 30–60 min at 37 °C. The adipocytes were separated from the stromal vascular cells (SVC) via centrifugation (200 rcf, 10 min, 4 °C) and later transferred into TRIzol^®-Reagent (Life Technologies GmbH, Darmstadt, Germany) for mRNA isolation, as described below. The purity of the adipocytes versus SVC after our isolation procedure was assessed by real-time PCR, using adiponectin for the adipocyte fraction, and using CD45 (indicating leukocyte lineage) for the SVC fraction of cells, as described in the Results section. The adiponectin was almost exclusively expressed in the adipocyte fraction of cells, indicating that we reached a high purity of adipocytes during our isolation procedure. The CD45, on the other hand, was almost exclusively expressed in the SVC fraction of cells, indicating that—as expected—this fraction contained a significant number of immune cells, which were absent in the adipocyte fraction of cells.

For primary cell culture, fresh intra-abdominal adipose tissue obtained from CTRP-3 KO mice was cut into small pieces and treated with 0.225 U/ml collagenase NB6 (Serva) at 37 °C for a maximum of 60 min. Digestion process was stopped by adding twice the amount of buffer (PBS containing 0.5% BSA and 2 mM EDTA). Cell suspension was filtered by 120-μm nylon mesh to eliminate undissolved tissue. Pre-adipocytes were separated from adipocytes by 10 min centrifugation at 300 g and 4 °C. Magnetic labeling plus depletion of non-adipocyte progenitor cells was done according to the manufacturer’s instructions (Adipose Tissue Progenitor Isolation Kit mouse, MACS Miltenyi Biotec, Bergisch Gladbach, Germany), as well as magnetic labeling and positive selection of adipocyte progenitor cells. Isolated pre-adipocytes were seeded at a density of 2.03 × 10⁴ cells/cm² in DMEM (Dulbecco’s Modified Eagle Medium, Biochrom AG, Berlin, Germany) that was supplemented with 10% newborn calf serum (NCS; from Sigma-Aldrich, Deisenhofen, Germany) and were cultured at 37 °C and 5% CO₂. Adipocyte differentiation was initiated after cells had reached 85 % confluency. Media for hormonal differentiation were supplemented as described above for 3T3-L1 cell line. Cellular phenotype during adipocyte differentiation was monitored by light microscopy.