The Fluo-4 Direct Calcium Assay was formulated to achieve the largest assay window by suppressing background fluorescence generated from media with little-to-no impact on the cellular fluorescence. This advanced formulation allows the assay to be run in a simple “addition only” format in the presence of serum-containing media.
Our Fluo-4 Direct assay can also be used with our large suite of validated GPCR cell lines to provide multiple read-out options for a single cell line. The Tango™ TBXA2R-bla U2OS Cell Line was used to multiplex the beta-arrestin recruitment response, as read out with the beta-lactamase reporter, with the direct measure of calcium mobilization using the Fluo-4 Direct assay (Figure 1).
Figure 1. The Fluo-4 Direct Calcium assay can be used to obtain calcium mobilization data to complement beta-arrestin recruitment data from Gq coupled receptors in our Tango offering.
Our Fluo-4 Direct assay can be used to obtain appropriate pharmacological profiles for compounds in both agonist (Figure 2) and antagonist (Figure 3) modes.
Figure 2.Dose-dependent calcium response to muscarnic 1 (M1) receptor agonists. CHO M1 cells were plated in a poly-d-lysine coated 384-well plate and incubated overnight. The following day, cells were assayed for a calcium response to carbachol using the Fluo-4 Direct assay. Cells were stimulated with the agonists carbachol, MCN-A-343, bethanechol, oxotremorine, and pilocarpine. Measurements are given in relative fluorescent units as the maximum response minus the minimum response divided by the minimum response. Rank order of agonist potency agreed with published results.