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.
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