*4.5. Adipocyte Di*ff*erentiation*

Exponentially growing ADSCs were collected, counted the number of cells, and 1 × 10<sup>5</sup> cells were replated onto 22 mm × 22 mm type I collagen-coated cover glass slips. They were cultured in a serum-free medium until they reached confluence. Then, the culture medium was changed to differentiation medium (DM-2, ZenBio, Inc., Research Triangle Park, NC, USA). They were cultured for another 10 days before fixation with 4% formalin. The fixed cells were stained with 10 μg/mL of BODIPY 493/503 (D-3922, Invitrogen, Carlsbad, CA, USA) for 20 min at room temperature and the nuclei were counterstained with 0.1 mg/mL of DAPI. Accumulation of lipid droplets was determined under a fluorescence microscope (F3000B, Leica, Tokyo, Japan). Digital images were captured and the images were analyzed by FW4000 software (Leica, Tokyo, Japan). Cells containing multiple lipid droplets in more than 50% of the cytoplasm were counted as differentiation positive cells. In order to quantify average fluorescence per cell, the same areas were marked, and the sum of the pixel intensity within the marked area was calculated by FW4000 software, and total green fluorescence was divided by total blue fluorescence obtained by DAPI staining.
