Nile red

Lipid droplet quantification by Nile Red staining was performed after incubation of cells with serum free medium for 5 days. Nile Red (15 μg/μL) (Santa Cruz Biotechnology, Dallas, United States) was added to the cell medium for 20 min at 37°C. Nile Red fluorescence was analyzed at 580 nm using an EnSpire Mulitmode plate reader (PerkinElmer, Waltham, United States). For normalization cell vitality was assessed using the alamarBlue assay (ThermoFisher, Waltham, United States) according to the manufacturer’s instructions.

The two types of piPSCs were digested, seeded 3×10³ cells per well into 96-well plate and detected at day 3. The mitochondria were stained by Mitotracker Deep Red FM (Life Technologies, M22426) according to the manufacturer’s instructions. The lipid droplets were stained using Nile Red (Sigma, N-1142). The cells were fixed with 4% PFA for 20 min at room temperature. Cells were washed three times with DPBS and then incubated with Nile Red solution (5 ng/ml) for 5 min at room temperature. To quantify the accumulation of cytoplasmic Nile Red and Mitotracker fluorescence in acquired images, the high content screening system “Operetta” was used in combination with the integrated image analysis software “Harmony” of Perkin–Elmer. From these images the intensity of Nile Red and Mitotracker fluorescence in the cytoplasmic area of each cell could be quantified by the software.