2.4. Nile Red Staining

Cells were seeded in 96-well plates and then subjected to the different treatments. After incubation, the neutral lipid accumulation was assessed through the Nile Red assay [30]. Briefly, the cell culture medium was discarded and 100 µL of the Nile red solution was added to each well for 1 h/1.5 h in the dark conditions at 37 °C. Nile Red is freshly diluted 1:200 in medium without FBS from the stock solution (stock: 0.5 mg/mL dissolved in acetone). Nile Red was then removed, and cells were washed twice with PBS 1X. The fat content per well (in 100 µL PBS 1X) was measured fluorimetrically with 520 nm excitation and 620 nm emission in a Biotek Cytation 3 reader (Biotek Instruments, Winooski, VT, USA). Results were normalized for cell mass content at the end of the assay, using the SRB method [31].