2.7. Determination of Lipid Accumulation, Free Fatty Acid Uptake and Triglyceride Content

The 3T3-L1 preadipocytes were seeded into a 96-well plate at a density of 10⁴ cells/well for each of the experiment. The lipid content in the mature adipocytes was determined using the Nile red staining method. After cell incubation with FJ and PJ, cells were washed with cold PBS and fixed in 5% paraformaldehyde for 30 min. Then, the cells were stained with Nile red (1 μg/mL) for 40 min and fluorescence intensity at F485/530 nm was measured. For cell nuclei visualization, to fixed cells, 1 μg/mL 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) stain was added. The measurement of fatty acid fluorescent probe TF2-C12 uptake by cells was performed with the Fatty Acid Uptake Kit (Sigma-Aldrich, Seinheim, Germany). After the cells’ treatment with the preparations, the fluorescent signal at F485/530 nm was measured after 1 h incubation with fluorescent analogue.

Triglyceride content was measured using the Triglyceride Colorimetric Assay kit (Cayman Chemical, Ann Arbor, MI, USA). To perform the experiment cells were seeded into a 6-well plate at a density of 2 × 10⁵ cells/well. Following treatment, the differentiated 3T3-L1 adipocytes were rinsed with PBS, harvested with a cell scraper, lysed with 1% Triton X-100 and the total triglyceride content was assessed according to the manufacturer’s instructions with an absorbance measurement at 540 nm.