Intracellular Calcium Dynamics Imaging

The levels of intracellular Ca²⁺ were determined using an LSM 510 confocal system from Carl Zeiss through computer-assisted video imaging on a single-cell basis. SH-SY5Y cells were treated onto 25 mm coverslips according to the experimental protocol. Following that, the cells were loaded with 4 µM Fluo-4/AM (Molecular Probe, Eugene, OR, USA) dissolved in Ca-PSS supplemented with 0.08% pluronic acid (Molecular Probe) for 40 min in darkness at RT. Cells were rinsed once with Na-PSS and subsequently treated with 1 μM thapsigargin in Na-PSS for 10 min [40 (link)]. By switching Na-PSS to K-PSS, we assessed the uptake of Ca²⁺ via the reverse mode. A peristaltic pump was used to change bath solutions, and images were captured every 5 s. A heated microscope stage and climate box from PeCon GmbH were employed to maintain the cells and perfusion solutions at 37 °C. An argon laser at 488 nm provided the excitation light, and the emission in the range of 505–530 nm was recorded in a time-lapse manner. Following image acquisition, we conducted the fluorescence intensity analysis offline, following the previously described methodology [40 (link)].