Ca2+-induced lysosome exocytosis assay

Lysosome exocytosis was induced in HeLa cells using the Ca²⁺ ionophore, ionomycin (Sigma-Aldrich), as previously described (Encarnação et al., 2016). Briefly, HeLa cells were incubated in Ca²⁺- and Mg²⁺-free ice-cold Hanks Balanced Salt Solution (HBSS, Gibco) with 2.5–10 µM ionomycin in the presence of 4 mM CaCl₂, for 10 min at 37°C. Cells incubated with HBSS alone were used as a control. After incubation, cells were placed on ice and processed for flow cytometry, β-hexosaminidase release assay or confocal cell microscopy.