Oil Red O staining assay

To visualize and measure of the lipid accumulation in preadipocytes, mature 3T3-L1 adipocytes and the effects of B. spergulifolius extracts on adipogenesis in 3T3-L1 adipocytes, was performed Oil Red O Staining Kit following the manufacturer’s instructions [20, 21]. The 3T3-L1 preadipocytes and adipocytes were cultured into a 24-well plate (2x10⁴ cells/well). After confluence, the medium was removed and cells were washed twice with PBS and fixed with 100 μL 10% (v/v) of formalin for 60 min at room temperature. The fixed cells were washed twice with PBS and then added with 100 μL 60% (v/v) of isopropanol for 5 min at room temperature. Oil Red O stock solution was prepared with 100% (v/v) isopropanol and was kept in 56°C water bath for 1 h. The Oil Red O working solution was prepared with 3-unit of Oil Red O stock solution to 2-unit water, and shook (Everlast Rocker 247, Benchmark, USA) for 10 min and filtered using Whatman no:1. The cells were incubated for 20 min at room temperature with working solution 100 μL. Then, the working solution was removed and wells were washed three times with water. The wells were added Hematoxylin for 1 min and then washed three times with water. Finally, cells were coated with water and images of them were taken under a microscope. Also, lipid accumulation in cells was quantified by eluting Oil Red O stain using isopropanol and optical density (OD) was measured at a wavelength of 490 nm using a spectrophotometer (Nanoprop 2000c, Thermo Scientific, USA) [22].