Intracellular Calcium Imaging with Fluo-3/AM

Calcium imaging was performed with Fluo-3/AM (Invitrogen, Carlsbad, CA, USA) as described previously (9 (link)). Briefly, the intracellular calcium levels were determined using a fluorescence microscope (ECLIPSE Ti-U; Nikon, Tokyo, Japan; and Leica DMi8 inverted microscope; Leica Microsystems Ltd., Wetzlar, Germany) with the calcium-specific fluorescent dye, Fluo-3/AM (5 µM; Invitrogen, Carlsbad, CA, USA). Fluo-3/AM was mixed with 0.1% F127 in normal buffer solution (NBS: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂/EDTA, 0.5 mM MgCl₂, 10 mM glucose, and 5.5 mM HEPES, adjusted to pH 7.4), and loaded into the cells by incubating for 60 min at 37°C. After 60 min of incubation, the cells were washed twice with NBS and replaced with fresh NBS. Following treatment with the compounds, the alterations in the fluorescent images were recorded on a computer connected to the microscope. The excitation and emission wavelengths were 488 nm and 515 nm, respectively. The intracellular calcium levels were expressed as the F/F₀ ratio, where F indicates the fluorescence intensity of the region of interest at a certain time point, and F₀ indicates the initial fluorescence intensity at 0 s. Image analysis was performed using the ImageJ software (NIH), with custom-made scripts for semi-automatic cell counting, calculation of the F/F₀ ratio, and image production.