Nile Red Lipid Quantification Assay

Nile Red Staining was performed as previously described (Benova et al., 2022 (link)). Cells were cultured in polystyrene flat-bottom 96-well tissue culture-treated black microplates (BRANDplates^®, cellGrade™, Brand, DE). Nile Red dye (Merck) working solution was prepared from a stock solution of 1 mg/ml. Cells were washed with PBS (Gibco). Dye was added directly to the cells (5 μg/ml in PBS), incubated for 10 min at room temperature in the dark, and then, washed twice with PBS. The fluorescent signal was measured using a Cytation 3 cell imaging multimode plate reader (BioTek) using an excitation of 485 nm and an emission of 572 nm. The fluorescent signal of Nile Red stain was normalized to cell viability signal measured by Cell Titer-Blue Assay Reagent (Promega) mentioned previously.