Daf fm da

Cell images using DIC, DAPI, FM4-64, DAF-FM DA, and Nile red staining were acquired using fluorescence microscopes [AxioPlan2 (Zeiss) and DeltaVision Elite (GE Healthcare)]. For nuclear staining, cells were fixed with 2% glutaraldehyde for 10 min on ice, washed three times with phosphate-buffered saline, and observed under a fluorescence microscope after mixing with DAPI (25 μg/ml) for staining. For vacuole staining, 1 μl of FM4-64 (1 μg/μl) (ThermoFisher) was added to 1 ml of cell culture, and cultures were incubated in the dark at room temperature for 45 min. After incubation, cells were washed in media without FM4-64, then incubated again for 45 min before observation. For nitric oxide staining, 5 μl of DAF-FM DA (1 μg/μl) (AdipoGen) was added to 1 ml of cell culture. Then, cultures were incubated in the dark at room temperature for 45 min. Cells were washed twice with media before observation. Signal intensity of DAF-FM DA was obtained using softWoRx software (GE Healthcare) and calculated by taking the average of 10 fluorescence points in each image. For lipid droplet staining, 1 μl of Nile red (1 μg/μl) (Wako) was added to 1 ml of cell culture, and cultures were incubated in the dark at room temperature for 5 min before observation.