Epididymal adipose tissues were cut into small pieces, followed by incubation in adipose isolation buffer (17 (link)) containing 1 mg/mL collagenase (Wako Pure Chemical Industries) for 1 hour at 37°C with gentle shaking. SVF cells were collected as a pellet by centrifugation at 500g and 4°C for 5 minutes. SVF cells were cultured in 10% FBS-DMEM supplemented with a penicillin/streptomycin solution (Thermo Fisher Scientific). Two days after reaching confluence, cells were incubated in differentiation medium (AdipoInducer Reagent [for animal cells]; Takara Bio Inc.) containing dexamethasone (2.5 μM), 3-isobutyl-1-methylxanthine (0.5 mM), and insulin (10 μg/mL) (included AdipoInducer Reagent [for animal cells]; Takara Bio Inc.) for 2 days. The medium was then replaced with maintenance medium (insulin [10 μg/mL] in 10% FBS-DMEM supplemented with antibiotics). The maintenance medium was renewed every 2 or 3 days for 6 days of culture. Adipocyte differentiation was evaluated by Oil Red O staining. Oil Red O uptake into cells was quantified by extraction with isopropanol, and the absorbance of the eluate was measured at 492 nm.
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