Lipid droplet detection with Nile red staining

Formation of lipid droplet in normal and oleate-induced cells was monitored using standard Nile red staining conditions as previously described [27]. Cells were grown on 2.4×2.4 cm cover glasses in 6-well plates in 2.5 ml cell culture medium. Following the determination of optimal oleate concentrations, oleic acid was added to each well at a final concentration of 20 μg/ml. Cells were cultured for an additional 24 h, 48 h, or 72 h. At the end of these time points, cover glasses were removed, washed 4–5 times with PBS and fixed with paraformaldehyde for 10 min. Diluted and filtered Nile red solution (1–2 ml, Sigma, USA) was added to the cover glasses and stained for 10 min at room temperature. Nile red solution was removed and, after washing 3–5 times with PBS, the cover glass was mounted on a glass slide and sealed. Confocal images were acquired using a laser scanning confocal microscope (Olympus, Japan). A total of 10 high-power fields (HPFs) were randomly selected and 3 cells per each HPF were randomly chosen for fluorescence intensity analysis using Olympus Fluview ver. 3 viewer software.