2.3. Oil red O staining and micrographs of lipid drop formation in 3T3-L1 cells

The lipid content of differentiated 3T3-L1 cells was evaluated by ORO staining on D2, D4, and D7 of differentiation. The 3T3-L1 preadipocytes were plated at a density of 2.5 × 10⁵ cells in a six-well plate and images were taken on D7. Cells were washed with phosphate-buffered saline (PBS) and fixed in 10% formalin in PBS for 1 h at 4°C, and then washed two times with PBS and stained with 0.5% ORO in 60% isopropanol for 1 h at 4°C. To quantify the intracellular lipid content, excess stain was removed by washing with 70% ethanol, and cells were extracted with 4% Nonidet P-40 (NP-40) in isopropanol. The absorbance of the extract solution was measured at 520 nm using a GENios microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Lipid drop accumulation in 3T3-L1 cells was photographed using an Olympus IX51 inverted microscope (Olympus Corp., Tokyo, Japan) at 100× magnification.