Differentiated SH-SY5Y cells were transfected with hippocalcin-mCherry constructs and loaded with 1 μM Fluo-4-AM (Molecular Probes) for 30 min in the absence or in the presence of 0.4 μM ω-agatoxin IVA (Abcam, P/Q-type calcium channel blocker), 10 μM ω-conotoxin MVIIC (Sigma, P/Q- and N-type calcium channel blocker) or 1 μM ω-conotoxin GVIA (Abcam, N-type calcium channel blocker). Cells were examined at 37 °C (OKO lab incubation chamber) in a 35-mm glass bottom dish (MatTek) with a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a 40x/1.3 oil immersion objective and a 3i Laserstack as excitation light source (488 nm, for Fluo-4; 561 nm, for hippocalcin-mCherry). Emitted light was collected through single bandpass filters (Yokogawa CSU-X filter wheel) onto a CMOS camera (Hamamatsu, ORCA Flash 4.0; 1152x1656 pixels).
Cells were stimulated with 50 mM KCl and images were collected every 5 s for 1 min. The time course for intracellular calcium influx was monitored over an elliptical region of interest (ROI) in the cell body using ImageJ program. Data obtained from 9 to 22 cells was plotted and analysed on GraphPad Prism.
Cells were stimulated with 50 mM KCl and images were collected every 5 s for 1 min. The time course for intracellular calcium influx was monitored over an elliptical region of interest (ROI) in the cell body using ImageJ program. Data obtained from 9 to 22 cells was plotted and analysed on GraphPad Prism.