Lysosomal Dynamics in DU145 Cells

DU145 cells were first incubated with DMSO or SB203580 (50 µM) for 24 h, followed by the addition of 2 µM LysoSensor™ Yellow/Blue DND-160 (Thermo Fisher Scientific, Cat # L7525) for 10 min. Cells were then evaluated at the MDACC Advanced Microscopy and Cell Imaging Core using a Zeiss LSM880 with Airyscan confocal microscope, using a Zeiss 40× c-apo (N.A. 1.2) water immersion lens; images were collected using an excitation wavelength of 355 nm (Coherent) and emission wavelengths in the yellow (510–641 nm) and blue (404–456 nm) channels, as previously described [72 (link)]. Image analysis was performed using Imaris (Andor/Oxford Instruments) software (version 9.9) Surfaces module; fluorescence intensities and lysosome/vacuole volumes were obtained for each and exported to GraphPad Prism software to calculate and graph their corresponding yellow/blue ratios.