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FLIPR Calcium 3 Assay Kit - An Optimal No-Wash Fluorescence Calcium Indicator Reagent for Non-Optimal Calcium Responses

ells were loaded with the calcium indicator dye FLUO-4AM reagent for 60 minutes at 37C. Subsequently, cells were washed once with HBSS at room temperature, 180 l HBSS was added to each well and plates were transferred to FLIPR for acquisition of calcium images. Excitation at 488 nm was achieved with an Argon ion laser and a 515 nm emission filter was used. In each experiment baseline fluorescence after dye loading was adjusted to 10,000 fluorescence counts by adjusting the strength of the laser. Fluorescence images and relative intensities were captured at 1 second intervals and cells were stimulated by addition of agonist (20 l) after 10 baseline measurements.

Calcium 3 FLIPR studies
Growth medium was removed by aspiration, 180 l Calcium 3 reagent, reconstituted in HBSS, was added and cells were loaded for 60 minutes at 37C. Subsequently, plates were transferred to FLIPR for acquisition of calcium images.


RESULTS
Calcium responses to a maximally effective concentration of 5-HT (10 M) in HEK cells transiently expressing the 5-HT2C (VSV isoform) receptor measured using either Calcium 3 or FLUO-4 are presented in Figure 1. In all data examples shown, cells were seeded at modest over-confluence at the time of transfection. That the results obtained for the 5-HT2C transfection are sub-optimal is evidenced by data from other experiments where maximum fluorescence counts in the range of 8,000-12,000 can be achieved with FLUO-4 using HEK cells transfected with the same construct under optimum cell seeding conditions. (See Figure 1, inset.) Figure 1 illustrates the advantage of using the Calcium 3 no-wash reagent under sub-optimal experimental conditions yielding a higher maximum signal in response to agonist, lower standard errors and improved well-to-well variability and elimination of the dilution artifact observed with FLUO-4 following agonist addition. In additio
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