Vb 7000 stereoscopic microscope

DFATs at P2 were seeded on 24-well plates at a density of 6 × 10⁴ cells per well and cultured in adipogenic differentiation medium (Mesenchymal Stem Cell Adipogenic Differentiation Medium 2: C-28016; PromoCell, Heidelberg, Germany). At 14 days of culture, samples were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 60 min. After fixation, samples were washed twice with PBS and stained with Oil red O staining solution (Sigma Aldrich) for 20 min. After being washed three times with distilled water, samples were photographed with a VB-7000 stereoscopic microscope (Keyence, Osaka, Japan). After the staining, samples were dried and incubated with 100 μl of isopropyl alcohol for 10 min. Then, the solution was collected and absorbance at 490 nm was measured with the microplate reader (iMark). Each quantitative value was evaluated from the average value of the absorbance at two wells to subtract the value of the blank containing only isopropyl alcohol. In another experiment, DFAT samples at 14 days of culture were used for real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis.

DFATs at P2 were seeded on 24-well plates at a density of 6 × 10⁴ cells per well and cultured in the osteogenic differentiation induction medium (Mesenchymal Stem Cell Osteogenic Differentiation Medium: C-28013; Promo Cell). At 14 days of culture, samples were washed twice with PBS and fixed with 4% PFA for 60 min. After fixation, samples were washed twice with PBS and stained with 1% Alizarin red S (Sigma Aldrich) for 3 min. After being washed three times with distilled water, samples were photographed with the VB-7000 stereoscopic microscope (Keyence). The intensity of Alizarin red S staining was quantified by modifying the method of Gregory et al. [26 (link)]. Briefly, 10% acetic acid was added to each well and incubated at room temperature for 30 min with shaking. Loosely attached cells were collected with a cell scraper and transferred to a 1.5-mL tube. After heating at 85 °C for 10 min, samples were centrifuged at 20,000×g for 15 min and the supernatant was collected. Then, 50 μl of 5% ammonia water (pH 4.1–4.5) was added, and the absorbance at 405 nm was measured with the microplate reader (iMark).