Quinacrine Visualization of Vesicular ATP

Quinacrine [4-N-(6-chloro-2-methoxyacridin-9-yl)-1-N,1-N-diethylpentane-1,4-diamine] was used to visualize vesicular ATP, a fluorescent dye widely used to detect ATP-enriched vesicles (Gutierrez-Martin et al., 2011 (link)). Quinacrine staining was carried out by incubating granule cells for 15 min at 37°C in Locke’s buffer (140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.5 mM glucose and 10 mM HEPES [pH 7.4]) containing 4 μM Quinacrine (Sigma–Aldrich). Time-lapse studies were performed with an Olympus IX81 inverted microscope equipped with a 100X, 1.45 NA, oil-immersion objective. Images were acquired every 200 ms with a Hamamatsu C9100 EM-CCD digital camera (Hamamatsu) controlled by CellR software (Olympus). Vesicular fusion and release into the extracellular space was observed as a rapid loss of the Quinacrine signal. During the assays, cells were continuously perfused with Locke’s buffer at a rate of 1.5 ml/min at 37°C. Perfusion was gravity-driven and solution exchange was performed by manually operating electronic valves of a VC-6 drug application system (Warner Instruments). Images were processed using the ImageJ free software v.1.50i (National Institutes of Health, Bethesda, MD, United States).