Indomethacin

DPSC and SHED were chemically induced to differentiate along chondrogenic, osteogenic, and adipogenic lineages by culturing monolayers of DPSC and SHED in specific differentiation medium for three weeks. Chondrogenic differentiation was induced using chondrogenic induction medium containing 20 ng Transforming growth factor beta-1 (TGFβ1) (Merck, Millipore), 10 ng insulin, 100 nM dexamethasone (Sigma-Aldrich, Inc., USA) and 100 μM ascorbic acid (Dae-Jung Chem and Metal Co, Korea). Cells were stained with 1% toluidine blue to detect extracellular matrix produced by chondrogenic derivatives. Osteogenic differentiation was induced by using osteogenic medium containing 0.1 μM dexamethasone (Serva, GmbH), 10 μM β-glycerophosphate (Sigma Chemical Corp, USA) and 50 μM ascorbate phosphate (Dae-Jung Chem and Metal Co, Korea). Cells were stained with Alizarin Red S stain (Sigma-Aldrich, Inc., USA) to visualize calcium deposition. Adipogenic differentiation was induced using adipogenic medium containing 0.5 μM isobutyl-methylxanthine (Sigma-Aldrich, Inc, USA), 1 μM dexamethasone (Serva, GmbH), 10 μM insulin (Sigma-Aldrich, Inc., USA), and 200 μM indomethacin (MP Biomedical). Cells were stained with Oil Red O (Sigma-Aldrich, Inc., USA) to determine presence of lipid droplets. Cells grown in regular media served as negative controls.

Lymphocytes Separation Medium, Luminol, Indomethacin as Hanks Balance Salts Solution and Lucigenin were obtained from MP Biomedicals Inc., Research Organics and Sigma ; Dimethylsulphoxide, ethanol and ammonium chloride of analytical grades from Merck Chemicals, Darmstadt, Germany; Zymosan A as Phorbol myristate acetate from Fluka and Human monocytic leukemia cells from European Collection of Cell Cultures; human TNF-α and IL-1β ELISA Kit from R&D systems, Minneapolis, USA and glass fiber filter and cell harvester from INOTECH, Dottikon, Switzerland.

The SVF cells were expanded in growth medium for approximately 4 days. The SVF cells were induced to differentiate into adipocytes as described before (26 (link)). In brief, the SVF cells at nearly 100% confluency were cultured in DMEM/F12 medium supplemented with 5% FBS, 1% ABAM, 2 mM L-glutamine, 17 nM insulin, 0.1 µM dexamethasone, 250 µM 3-Isobutyl-1-methylxanthine (IBMX), and 60 µM indomethacin (MP Biomedical, Solon, OH), for 2 days. insulin, dexamethasone, and IBMX were all purchased from Sigma-Aldrich. The SVF cells were then cultured in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, 1% ABAM, and 17 nM insulin for 2 days. Lastly, the SVF cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, and 1% ABAM, for 4 days. To determine the effect of GH on lipolysis, the SVF cells on the 8^th day of differentiation were treated with 100 ng/ml recombinant bovine GH or control in serum- and insulin-free medium for 4 h and 24 h, and medium samples were taken for glycerol assay.

The drugs were purchased from Sigma Chemical Co. St. Louis, MO, USA (N-ethylmaleimide, omeprazole, Nω-nitro-L-arginine methyl ester hydrochloride, and ethanol), MP Biomedicals (indomethacin), and Merck (organic solvents).

To induce adipogenic differentiation, BMSCs at the 1^st passage were cultured in adipogenesis-inducing media containing 0.5 mM isobutylmethylxanthine (MP Biomedicals, Irvine, CA, USA), 0.5 μM DEX (Sigma-Aldrich, St. Louis., MO, USA) and 60 mM indomethacin (MP Biomedicals, Irvine, CA, USA). Cells were plated at 2 × 10⁵ cells/well in 12-well plates. The media were changed every 3 days. After induction for 14 days, oil red O staining was performed to determine the lipid droplet formation in adipogenesis1 (link). Quantitative parameters of the percentage of the area, the number and the average size of lipid droplets were determined from at least 5 fields of view with the ImageJ 1.47 software. The quantification was performed based on semi-automatic plug-ins for recording and repeating same operation steps. The thresholds for distinguishing the quantification objectives with the background were set based on preliminary tests with several differential photographs.

For analysis of adipogenic differentiation, the cells (2 × 10⁴ cells/cm²) were sown on Petri dishes (Corning, Corning, NY, USA), covered with 0.1% gelatin (Sigma, Steinheim, Germany), and grown to 90% confluence. The cells were then incubated for 5 weeks in a medium composed of 10% FCS, 10 μg/mL insulin (Sigma, Steinheim, Germany), 1 μM dexamethasone (Sigma), 250 μM 3-isobutyl-1-methylxanthine (Sigma), and 200 μM indomethacin (MP Biomedicals, Solon, OH, USA). The medium was changed every 3 days. To detect fat deposition, the cells were fixed with 10% formaldehyde for 30 min. Fat drops were stained with Oil Red dye (Sigma, USA) according to the protocol of the manufacturer. Images were taken at 200× magnification. Control cells were cultured in growth medium without the addition of stimulating factors.