Ix50 s8f2

To confirm adipocyte differentiation, the hADMSCs were seeded in 12-well plates. Differentiation was initiated when confluence was 80%. The corresponding stains were carried out at day 14 of differentiation. The cells were washed twice with PBS and then fixed with 10% paraformaldehyde for 30 minutes at room temperature. Finally, two washes were performed with PBS, and Oil Red O (Trevigen cat. number 5010-024-05) prepared according to the supplier was added followed by incubation for 30 minutes with gentle shaking and protection from light. Two additional washes were then carried out, and PBS was added for the inverted microscope display (Olympus IX50-S8F2). For quantification, the PBS was then removed, and the dye was extracted with isopropanol and incubated for 10 minutes with shaking and protection from light. The supernatant was finally placed in a quartz cell (Quartz Spectrophotometer Cell Semi Micro, 9-Q-10 mm, Bio-Rad Laboratories), and the absorbance of the sample was measured on a spectrophotometer (Ultrospec 3000, Pharmacia Biotech) at 500 nm using isopropanol as a blank to obtain the relative lipid accumulation.

To confirm adipocyte differentiation, the hASCs were seeded in 12-well plates. Differentiation was initiated when confluence was 80%. The corresponding stains were carried out at days 0, 7, and 14 of differentiation. Cells were washed twice with PBS and then fixed with 10% paraformaldehyde for 30 min at room temperature (RT). After that, 2 washes with PBS were performed, and Oil Red O (Trevigen Cat. No. 5010-024-05) prepared according to the supplier was added, followed by incubation for 30 min with gentle shaking and light protection. Finally, PBS washes were performed twice, and cells were observed at an inverted microscope (Olympus IX50-S8F2) to acquire images with magnifications of 4x and 40x. For stain quantitation, the PBS was removed, and dye was extracted with isopropanol (750 μL) and incubated for 10 min with shaking and light protection. The supernatant was collected, and absorbance of the sample was measured on a spectrophotometer (Ultraspec 3000), Pharmacia Biotech) at 500 nm using isopropanol as a blank to obtain the relative lipid accumulation.