Measuring Intracellular Calcium Dynamics

Intracellular calcium assay was performed as previously described [30 (link)]. The culture medium of the Flp-In NCC HEK293 cells was replaced by loading buffer containing 5 μg/ml Fluo 4-AM (Dojindo, Kumamoto, Japan), 1.25 mmol/l probenecid (Dojindo, Kumamoto, Japan), and 0.02% Pluronic F-127 (Dojindo, Kumamoto, Japan). Following incubation with 5 mM EGTA or 1 μM SEA0400 in the loading buffer for 1h at 37°C, the loading buffer was replaced by recording medium containing 1.25 mmol/l probenecid. NCX1 siRNA silencing was applied 48 h before loading buffer replacement. Following the administration of KCl (final concentration: 10 mM), the fluorescence intensities of Fluo 4 were quantified from five regions of interest using LSM 510 Meta confocal microscopy and the Zen 2009 software (Carl Zeiss, Oberkochen, Germany).
http://dx.doi.org/10.17504/protocols.io.baihicb6