2.1.2 Calcium imaging:
Cells were pre-incubated with a normal Ringer’s solution (in mM: NaCl
14, HEPES 10, KCl 5.4, MgCl2 0.5, CaCl21.8 and glucose 5), including Fluo-4 (2.5µM; 45min) as the fluorescent
Ca2+ indicator. Following the loading process, the
cells were washed with a normal Ringer’s solution to ensure that the
excess Fluo-4 AM was removed. The coverslips were transferred to the
perfusion chamber constantly supplied with Ringer’s solution through the
perfusion system. The whole experiment was performed in a darkened room
to keep the background illumination at the lowest level and prevent
bleaching of the fluorophore.
The increase in fluorescence associated with Ca2+binding can be monitored using an excitation wavelength of 488nm with
detection at > 500 nm. Images were taken using an Olympus
inverted microscope with a 10x lens and an Olympus fluoview 300 (version
4.2) software. At the end of each experiment, the cells were challenged
with a 60 mM KCl Ringer’s solution to confirm the viability of the cells
because a High-K + Ringer’s solution induces
depolarization only in living cells. The change in fluorescence was
normalized as F510 self-ratio (F510 S-R) by this equation
\begin{equation}
F510\ S-R=\frac{fluorence\ intensity-background\ intensity}{baseline\ intensity-background\ intensity}\nonumber \\
\end{equation}For Ca2+ response experiments, all raw data were
transferred from the image J into Excel sheets for primary calculation,
followed by transfer them to GraphPad and analyzing them using GraphPad
Prism version 6.00 (GraphPad Software, LaJolla, California, USA).