Quantifying Intracellular Lipid Droplets

Nile Red Staining was performed as we previously described [20 (link)]. It is a direct stain for the detection of intracellular lipid droplets by fluorescence microscopy. Cells were cultured in polystyrene flat-bottom 96-well tissue culture-treated black microplates (BRANDplates®, cellGrade™, Brand, Wertheim, Germany). Nile Red dye (Merck, Darmstadt, Germany) working solution was prepared from a stock solution of 1 mg/ml. Cells were washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA). Dye was added directly to the cells (5 μg/ml in PBS), and incubated for 10 min at room temperature in the dark, then washed twice with PBS. Fluorescent signal was measured using a Cytation 3 cell imaging multimode plate reader (BioTek, Winooski, VT, USA) using excitation of 485 nm and 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, Madison, WI, USA) mentioned above.