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

gative control. The term |c+ c- | denotes the absolute value of the difference between the mean of the positive control and the mean of the negative control. A Z-factor > 0.5 indicates a large separation band between the negative and positive controls and an excellent assay.


RESULTS

ATP elicited a concentration-dependent increase in [Ca2+]i in CHO cells (Figure 1) with similar EC50 values for cells loaded with Fluo-3, Fura-2 and FlexStation Calcium Assay Kit, respectively (Table 2).

One-way Analysis of Variance (ANOVA) indicated that the EC50 estimates were not significantly different from one another (P value = 0.62).


These results indicate that there is negligible difference in the ATP EC50 values from cells loaded with the three fluorescent reagents; however, there was a significant difference between the peak fluorescent intensities of the two single-wavelength indicators. With Fluo-3 there was an increase in signal of ~3,200 RFU above basal (1.8 fold increase) whilst with the FlexStation Calcium Assay Kit the increase in signal was 32,000 RFU (5.6 fold increase, see Table 1). Peak response tended to be more variable with Fluo-3 (see Figure 1). With Fura-2 there was a 4.8 fold increase in signal above basal.

The screening coefficient window (Z-factor), which reflects the dynamic range of the signal and the data variation for the assay9 was calculated using the buffer addition (negative control) and the 30 M ATP addition data (positive control). Z-factors obtained with Fura-2 and the FlexStation Calcium Assay Kit were 0.60 and 0.68 respectively (Table 1). This shows a large separation band between the negative and positive controls and a reproducible, high quality assay. The Zfactor obtained for the Fluo-3 data was 0.28, representing a useable assay.
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