Lysosome Calcium Measurement with GCaMP3-ML1

Lysosome calcium measurement was performed using methods described previously [10 (link)]. Briefly, 2 × 10⁵ HT1080 cells stably expressing GCaMP3-ML1 were cultured in a 35-mm confocal dish (SPL Life Sciences, 100350). Changes in cytosolic Ca²⁺ levels were monitored by following changes in GCaMP3-ML1 fluorescence for 15 min upon addition of 200 μM GPN in Ca²⁺-free external solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA (Sigma-Aldrich, E3889), 20 mM HEPES (Gibco, 15630080), pH 7.4, using the real-time mode of epifluorescence microscopy (Applied Precision DeltaVision Elite, Applied Precision Inc., USA). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of GCaMP3-ML1 fluorescence, and the mean fluorescence intensity was monitored at 488 nm. The acquired epifluorescence images were numerically deconvolved using DeltaVision algorithms (Applied Precision Inc., USA).