Og bapta am

HT1080 cells were cultured in a 35-mm confocal disc in a CO₂ incubator at 37°C for 24 h. After overnight culture, cells were loaded with OG-BAPTA-AM (Thermo Fisher Scientific, O6807) in the culture medium at 37°C for 1 h. OG-BAPTA-AM-loaded cells were washed thrice with Ca²⁺-free external solution and incubated at 37°C in an imaging chamber for 10 min. The release of lysosomal Ca²⁺ was monitored by following changes in OG-BAPTA-AM fluorescence (494 nm/523 nm) over 15 min upon the addition of 200 μM GPN using real-time mode. Ca²⁺ responses were recorded using a DeltaVision microscope (Applied Precision Inc., USA). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of OG-BAPTA-AM fluorescence at 488 nm, and the mean fluorescence intensity was calculated. The acquired epifluorescence images were numerically deconvolved using DeltaVision algorithms.