Oil red o powder

To prepare the oil red O stock solution, 500 mg of oil red O powder (Nacalai Tesque, Kyoto, Japan) was dissolved in 100 mL of 99% isopropanol. Theoil red O working solution was prepared just before use by diluting 60 mL of the stock solution with 40 mL of distilled water. To measure lipid accumulation, the cells were washed with phosphate-buffered saline (PBS), fixed with 10% formalin for 15 min, rinsed with 60% isopropanol, and stained with oil red O working solution for 15 min at 37 °C, followed by washing with PBS. Brightfield images were taken with identical exposures on a Nikon DS-L2 camera. Oil red O was extracted with 99% isopropanol and the absorbance was measured at 490 nm using Microplate Reader SH-1000Lab (Corona, Ibaragi, Japan). Empty wells stained with oil red O working solution were used as background, and absorbance was subtracted from each sample for quantification.

Geltrex and basic fibroblast growth factor (bFGF) were purchased from Life Technologies (Carlsbad, CA, USA). The CytoTune-iPS ver. 1.0 Sendai Reprogramming Kit was purchased from DNAVEC (Ibaraki, Japan). Oil Red O powder was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Percoll Plus was purchased from GE Healthcare UK (Buckinghamshire, England). The adipogenic stimulation cocktail ingredients insulin, IBMX, and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, USA).