2.4. Nile red staining

RAW264.7 cells were fixed with 4% paraformaldehyde for 40 minutes and then washed with PBS three times. Nuclei were stained with DAPI (2.5 μg/ml, in methanol) for 15 minutes at 37°C followed by washing with methanol. Then, the cells were stained with Nile red (10 μg/ml, in methanol) for 0.5 hours at 37°C. After extensive washing with PBS, confocal fluorescence images were obtained using a confocal microscope (Zeiss, Germany). Cellular lipid content was measured by determining the integrated density ratio (Nile red/DAPI).