Nile red staining

Live cells seeded on cover glasses were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and then incubated with Nile red (HY-D0718, MCE, USA) at 1:2000 in phosphate-buffered saline (PBS) for 10 min. The slides were counterstained with Hoechst 33342 (H1399, Thermo Fisher, USA) at 1 μg/ml in PBS for 5 min at RT before imaging. The cells were visualized with a fluorescence microscope (Olympus, Tokyo, Japan). A representative image is shown from three independent experiments.

Fluorescence emitted by cells stained by Nile red was measured at 595 nm by flow cytometry (BD Biosciences, USA), during the assay 10,000 cells per sample were collect and count. The result analyzed with Cell Quest software (BD Biosciences, USA) and expressed as mean of fluorescence intensity (MFI).