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Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation

This probably reflects the less than optimal assay protocol and subsequent small dynamic range of the assay.


SUMMARY
In this study, ATP caused a concentration-dependent increase in [Ca2+]i in CHO cells endogenously expressing the P2Y2-receptor, with a potency similar to that reported previously. Furthermore, we have demonstrated that the FlexStation can be used to generate reliable, reproducible data for calcium mobilization assays used to monitor such Gq-coupled GPCRs.

The EC50 estimates for ATP were similar, whether the fluorescent dye Fluo-3 (0.639uM) or Fura-2 (0.639uM) or the FlexStation Calcium Assay Kit (0.651uM) was used, and were consistent between assay plates on the same day, as well as between assays analyzed on different days (less than 0.6 log unit differences). There were, however, significant differences in the dynamic ranges from the three different loading protocols, which is reflected in the corresponding Z-factors. Less than a twofold increase in signal above basal was observed when the cells were loaded with Fluo-3. The most likely cause is the subsequent wash steps in the protocol, reducing cell responsiveness. Using cells of a lower passage number and a dedicated cell-washer (Denley or similar) would probably have improved the Z-factor for Fluo-3 loaded cells. However, under these experimental conditions there was a 5-fold increase in signal observed with Fura-2, despite the cells undergoing a number of wash steps. This could be attributed to the beneficial ratiometric properties of the dye. However, the biggest fold increase in signal and Z-factor was observed with the FlexStation Calcium Assay Kit indicating that this "no-wash", homogeneous assay can lead to stronger signals, higher assay precision and, thus, improved physiological data.

In conclusion, FlexStation provides an easy to use, ver
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