Dulbecco s modified eagle medium

Human U87 and U251 malignant glioblastoma multiforme cells were obtained from ATCC/LGC-2397 (Wesel, Germany). Cells were cultured in Dulbecco's modified Eagle medium (Biochrom, Berlin, Germany) containing 10% fetal bovine serum (Biochrom, Berlin, Germany) with 100 U/ml penicillin, 100 μg/ml streptomycin (Biochrom, Berlin, Germany) and 1% L-glutamine (Gibco/Invitrogen, California, USA). Cells were cultured at 37°C with 5% CO₂ and saturated humidify.

After centrifugation, the collected liquid PRF from each protocol was transferred into a cell culture plate. The plate was placed in 37 °C degree incubator until all the samples formed a clot. Afterwards, Dulbecco’s Modified Eagle Medium (Biochrom GmbH, Berlin, Germany) was added to all clots and further incubated in 37 °C degree to allow growth factor release. The supernatant (5 ml) was collected after 1 h and frozen. The collected supernatant was replaced by a fresh cell media and further incubated for 24 h. At the latter time point, the supernatants were collected. The collected samples at both time points were analyzed for human vascular epithelial growth factor (VEGF) and human transforming growth factor (TGF-β1). Protein quantification was performed by means of ELISA (DuoSet^® ELISA RND system) according to the manufacturer’s instructions. Optical density was assessed using a microplate reader at 450 and 570 nm. The data measured at 570 nm were subtracted from the data measured at 450 nm for an optical density correction. The measurements were performed in triplicates for each protocol and donor. Finally, the quantified data were statistically analyzed.

The 3T3-L1 preadipocytes were ordered from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated at 37 °C and 5% CO₂ in Dulbecco’s Modified Eagle Medium (Biochrom, Berlin, Germany) supplemented with 10% newborn calf serum (Sigma Bioscience, Deisenhofen, Germany) and 1% penicillin/streptomycin (PAN, Aidenbach, Germany). For adipogenesis, 3T3-L1 preadipocytes were grown to confluence and differentiated into adipocytes as described (Bauer et al. 2011 (link)). To study the effect of inflammatory factors during differentiation, the substances were added at day 0 (start of differentiation) and medium was changed at day 3, 6, 7 and 8 during differentiation.

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.

Four human HCC cell lines, Hep3B, HepG2, PLC, and HuH7 were used. Hep3B, HepG2, and PLC had been obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), whereas HUH7 had been obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Japan). HuH7, Hep3B, and PLC were grown in Dulbecco’s Modified Eagle Medium (Biochrom, Berlin, Germany), whereas HepG2 was maintained in RPMI 1640 medium (Biochrom, Berlin, Germany). Both media were supplemented with 10% heat-inactivated fetal bovine serum (Biochrom, Berlin, Germany) and 1% penicillin-streptomycin (Invitrogen, Darmstadt, Germany). The cells were stored lying flat in 175 cm² tissue plastic flasks (Falcon, Becton-Dickinson Labware Europe, Le Pont de Claix, France) in an incubator at 37°C in humidified air with 5% CO₂ and passaged weekly.