A TIRF microscopy-based assay to monitor LRRK2-dependent endo-lysosome exocytosis

Here we describe a method for monitoring CD63+ endo-lysosome exocytosis in hTERT-RPE cells stably expressing CD63-pHluorin. The fluorescence emission of pHluorin, a pH‑sensitive fluorescent probe, is quenched within acidic compartments such as lysosomes and multi-vesicular bodies (Miesenböck et al., 1998; Verweij et al., 2018). When these compartments fuse with the plasma membrane, the sudden shift in pH from acidic to neutral causes the probe to fluoresce. CD63-pHluorin enables monitoring of fusion events at the plasma membrane using Total internal reflection fluorescence (TIRF) microscopy. Powerful ExoJ software (Liu et al., 2024) enables automated quantitation of individual events. This protocol includes siRNA transfection to monitor the effects of specific protein depletion on endo-lysosome exocytosis events.