Fluo-4 Direct™ Calcium Assay Kit

  • Compatible with media containing serum - no removal of serum containing media necessary
  • Homogeneous add-and-read format – load cells, stimulate and read
  • Large assay window – low background with high fluorescence intensity

For more information about the Fluo-4 Direct™ Calcium Assay Kit, email us at Fluo4Direct@Invitrogen.com.

Fluo-4 Direct™ Calcium Assay Kit in addition-only format

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.

Complement other readout options

Invitrogen’s Fluo-4 Direct™ assay can also be used with Invitrogen’s 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 Invitrogen’s Tango™ offering.

High quality data

Invitrogen's 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.

Figure 3. Dose-dependent inhibition of the calcium response by M1 receptor antagonists. 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. The ability of antagonists scopolamine, telenzipine, and DAMP to block the calcium response elicited by 114 nM carbachol (EC80 for this receptor) was tested. Measurements are given in relative fluorescent units as the maximum response minus the minimum response divided by the minimum response. Rank order of antagonist potency agreed with published results.
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