Figures 2-4 present results from similar comparisons between Calcium 3 and FLUO-4 for the 5-HT1A, 5-HT6 and 5-HT7 receptors transiently expressed in HEK cells. Calcium signaling was facilitated with the 5-HT1A and 5HT6 receptors by co-expression with a chimeric G-protein, and the 5-HT7 receptor was coexpressed with a promiscuous G-protein. Under optimal experimental conditions responses in the range 6000-7000, 5000-6000 and 2000-4000 have been measured with FLUO-4 for 5-HT1A (Figure 2, inset), 5-HT6 and 5-HT7 (not shown) transient expression, respectively. In the 5-HT1A example (Figure 2), use of Calcium 3 resulted in an improved assay performance similar to that described above for the 5-HT2C receptor, i.e., specifically enhanced signal, less variability and reduced dilution artifact. Under optimal conditions, Calcium 3 and FLUO-4 exhibited equivalent performance both in terms of signal range and variability (Figure 2, inset). In the case of the 5-HT6 (Figure 3) and 5-HT7 (Figure 4) receptor evaluations, the difference in assay performance attributable to use of Calcium 3 following non-optimal cell seeding conditions was much more dramatic. Robust calcium transients were measured in the assays run with Calcium 3, while profiles obtained with FLUO-4 were subject to a large dilution artifact and small signal range in response to agonist stimulation.
A number of fluorescent indicator dyes, including FURA-2, FLUO-3, FLUO-4 and the no-wash reagent FLIPR Calcium and Calcium Plus assay kits, are available for the measurement of