Adipogenic Differentiation of CD146+ Pericytes

CD146⁺ adipose pericytes from 3 to 8 passages were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and allowed to adhere overnight. At 24 hours after seeding, the basal medium was replaced with adipogenic differentiation medium and replenished every 3 days (Mesencult Adipogenic Differentiation medium, StemCell Technologies Inc.). Oil Red O staining was performed after 10 days of differentiation (4 (link), 40 (link), 42 (link)). Cells were washed with PBS and fixed with 4% formaldehyde for 15 minutes. After fixation, cells were washed with water and 500 μL of Oil Red O staining solution. Oil Red O stock solution was prepared by dissolving 0.5 g of Oil Red O in 100 mL isopropanol. Oil Red O staining solution was prepared by dilution of a stock solution with distilled water in a 3:2 ratio, followed by filtration. Oil Red O staining was performed for 30 minutes at 37°C. Following incubation, cells were washed with tap water, followed by microscopy. After imaging, Oil Red O stain was extracted with 100% isopropanol for 5 minutes followed by an absorbance at 548 nm for quantification. All experiments were performed with n = 3 human samples per anatomic depot and in triplicate wells (biologic and technical triplicate).