1 methyl 3 isobutylxanthine ibmx

Adipose differentiation was induced by seeding 5 × 10³/cm² of f-LSCs and culturing in home-made differentiation medium consisting of DMEM medium containing 10 % FBS, 0.5 nM 1-methyl-3-isobutylxanthine (IBMX; Sigma-Aldrich), 10^–4 mM dexamethasone, 10 μg/ml insulin, and 100 μM indomethacin (Sigma-Aldrich) [34 (link)]. To detect the presence of lipid droplets, cells were fixed with 4 % formaldehyde solution, rinsed twice in distilled water, stained with Oil Red O (Sigma-Aldrich), and observed under a light microscope at 20–40× magnification.

RAW 264.7 cells and 3 T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco’s modification of Eagle’s medium (DMEM) was purchased from Gibco® by Life Technologies Co. (Carlsbad, CA) and Fetal bovine serum (FBS) from Hyclone (Logan, UT). Assay kits of total cholesterol, triglyceride, HDL-cholesterol, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glutamic pyruvate transaminase (GPT), Gamma-glutamyl transferase (γ-GTP), Blood urea nitrogen (BUN), and Creatinine were purchased from Asan Pharm Co., Ltd (Whasung, Korea). (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dexamethasone (DEX), insulin, 1-methyl-3-isobutylxanthine (IBMX), Nicotinamide adenine dinucleotide phosphate (NADPH), Oil red O, Xanthine oxidase, and Hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO).

Adipogenic differentiation of ASCs was performed as previously described [24 (link)]. Subcutaneous and bladder-associated ASCs plated in the growth media were cultured until confluence in a 35 mm dish. The medium was removed, and the cells were incubated with or without the adipogenic differentiation medium consisting of the DMEM/F12 medium supplemented with 10% FBS with the addition of 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO), 10 µg/ml recombinant human insulin, and 0.5 mM 1-methyl-3-isobutyl xanthine (IBMX) (Sigma-Aldrich) for three days. This medium was replaced every three days with the same medium without IBMX for the adipogenic differentiation maintenance period. After 15 days of differentiation, stromal and adipogenic cells were fixed in 1% paraformaldehyde in PBS and stained using the lipid stain Oil Red O.

For the induction of adipogenesis, the ASCs were seeded at a density of 50,000 cells/cm² in 6-well cell culture plates. Adipogenesis was induced using differentiation medium, composed of 0.2 μM insulin (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM 1-methyl-3-isobutylxanthine (IBMX) (Sigma-Aldrich, St. Louis, MO, USA), 0.25 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 10 μg/mL transferrin (Sigma-Aldrich, St. Louis, MO, USA) in ASC medium. After 3 days of differentiation, the medium was changed, and the cells were cultivated in the differentiation medium without IBMX for 14 days.

For osteoblast differentiation, the cells were cultured in osteoblastic induction media (OIM) comprised of basal media supplemented with 10 mM β-glycerophosphate (Calbiochem-Merck, Darmstadt, Germany), 50 μg/ml L-ascorbic acid-2-phosphate (Wako Chemicals GmbH, Neuss, Germany), 10 nM dexamethasone (Sigma-Aldrich, Brøndby, Denmark), and 10 nM calcitriol (1.25-dihydroxy vitamin-D₃ (1,25 (OH)₂D₃) kindly provided by Leo Pharma, Ballerup, Denmark). For adipocyte differentiation, the cells were cultured in adipocytic induction media (AIM) containing basal media supplemented with 10 % horse serum (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), 500 μM 1-methyl-3-isobutylxanthine (IBMX) (Sigma-Aldrich), 1 μM Rosiglitazone (BRL49653; Cayman Chemical, Ann Arbor, MI, USA), and 5 μg/ml insulin (Sigma-Aldrich). Samples undergoing induction were collected at days 5, 10, and 15. Three independent experiments were performed for each differentiation assay.

To initiate adipogenesis, ASCs were seeded at a density of 50,000 cells/cm² in 6-well cell culture plates and allowed to reach confluence. The induction of adipogenesis was carried out using a differentiation medium consisting of 0.2 μM insulin, 0.5 mM 1-methyl-3-isobutylxanthine (IBMX), 0.25 μM dexamethasone, and 10 μg/mL transferrin, all obtained from Sigma-Aldrich, St. Louis, MO, USA. This differentiation medium was prepared by supplementing ASC medium. After 3 days of differentiation, the medium was replaced, and the cells were cultured in differentiation medium without IBMX for 14 days.

The mouse preadipocyte cell line (3T3-L1) was obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) containing 10% bovine calf serum (BCS; Gaithersburg, MD, USA) and 1% penicillin/streptomycin antibiotics (P/S; Gaithersburg, MD, USA). To induce adipogenesis, 3T3-L1 preadipocytes (4 × 10⁴ cells/well) were grown in a 24-well plate for 2 days, and then the culture medium was replaced with the adipogenic differentiation medium containing 0.4 μg/mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS; Gaithersburg, MD, USA), 1-methyl-3-isobutylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO, USA), 1% P/S antibiotics, and 5 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 days, the culture medium was replaced with DMEM supplemented with 10% FBS, 5 µg/mL insulin, and 1% P/S antibiotics every 2 days. Finally, the culture medium was replaced with DMEM containing 10% FBS and 1% P/S antibiotics, which was changed every 2 days, as previously described [50 ]. Hispidulin (5, 10, 20, and 40 μM) and p-synephrine (5, 10, 20, and 40 μM) were added individually or in combination in the culture medium during adipogenic differentiation. Hispidulin (≥98%) and p-synephrine (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).