Quantifying Acidic Endosome Fluorescence

The distribution and fluorescence intensity of acidic endosomes in the cells were measured with LysoSensor DND-189 dye (Molecular Probes, Eugene, OR, United States) using live-cell imaging (Lusamba Kalonji et al., 2015 (link)). The cells on coverslips in Petri dishes were observed under a fluorescence microscope (Olympus IX70; Olympus Co., Ltd., Tokyo, Japan). The excitation wavelength was 443 nm, and the light emitted from the cells was detected using a 505 nm filter. Fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani Co. Ltd., Fukui, Japan) equipped with a fluorescence microscope. HNE cells were treated with kakkonto (20 μg/ml) or media alone. The fluorescence intensity of the acidic endosomes was measured in 100 cells, and the mean value of the fluorescence intensity was expressed as a percentage of the control value compared to the fluorescence intensity of the cells prior to treatment.