2.5.1. Oil Red O Staining

Oil Red O is a fat-soluble dye that stains neutral triglycerides and lipids [27]. Intracellular triglyceride droplets of hADMSCs were stained with Oil Red O solution on day 4, 10, and 21. The sample preparation procedure was as follows: medium was removed, each well was rinsed twice in PBS, and fixed in 4% formaldehyde for 1 h at room temperature. The cells were then rinsed twice with distilled water and stained with Oil Red O solution (0.5 g of Oil Red O powder dissolved in 100 mL of isopropanol). This solution was blended with distilled water in the ratio 3:2. Cells were incubated with this solution for 15 min at room temperature, then washed twice with distilled water and visualized using the Olympus CX41 microscope (Olympus, Tokyo, Japan) connected to a digital camera.

Figure 3 shows the intracellular fat droplets during differentiation in all four types of the media.

The comparison of fat in each environment. The arrows show examples of fat droplets. (Oil Red O staining).