2.3.6. Oil Red’O and Nile Red Staining Analysis to Determine Lipid Accumulation Levels

Differentiated preadipocytes were maintained in 24-well plates and treated with ZSE, AgNO₃, ZS-Ag-NPs (0.1, 0.2 and 0.4 µg/dL), and orlistat (6 µM) and incubated in the CO₂ incubator for 14 days. The maintenance media were changed once in 3 days. After 14 days, cells were washed twice with PBS and fixed with 4% (v/v) paraformaldehyde for 1 h at room temperature. Thereafter, the treated cells were subsequently washed with PBS and isopropanol 60% (v/v) and left to dry. Then, the treated cells were stained with a filtered 0.5% (v/v) Oil red’O solution (60% isopropanol and 40% water) for 1 h. Then, the Oil red’O staining solution was removed, and the plates were rinsed with distilled water thrice and left to dry. The stained lipid droplets were viewed at 20× magnification on a microscope and were photographed. After the image analysis, the stained cells were dried overnight and the oil stains were dissolved with isopropanol to measure the absorbance at 520 nm.

For the Nile red staining assay, a stock solution containing 5 mg of Nile red dissolved in 1 mL of 100% acetone was used. After 14 days of ZSE, AgNO₃ and ZS-Ag-NPs (0.4 µg/dL) treatments, preadipocytes were fixed with formaldehyde, then stained with 200 μL of fluorescence Nile red (working solution: 6 μL of stock Nile red dissolved in 1 mL of 40% isopropanol) for 30 min at room temperature. Then, the stained cells were analyzed using an inverted fluorescence microscope and photographs were taken immediately using a fluorescent microscope.