• Proven pharmacology—most rigorously tested, highly cited and used dye in the industry.
  • Robust—provides high and consistent Z’-factors with reduced washing steps (Table 1)
  • Flexible—allows screening with both adherent and non-adherent cell lines
  • Coupled assays—combine with our validated GeneBLAzer or Tango™ GPCR cell lines

We have many fluorescent calcium indicators for various applications that do not use quenchers, but the two which are based on the market leading Fluo-4 core chemistry are Fluo-4 AM and Fluo-4 NW.

Fluo-4 AM is an improved analog of Fluo-3 AM with the two chlorine substituents replaced by fluorines . This minor structural modification results in a dye that loads faster and is brighter at equivalent concentrations, making it the preferred indicator for a variety of applications: microplate screening (Figures 1 and 2), confocal microscopy (Figure 3), and flow cytometry.

The Fluo-4 NW Calcium Assay provides the same advantages as Fluo-4 AM but with the added convenience of an improved formulation that includes PowerLoad Concentrate, an improved dye loading reagent, and eliminates the need for washing steps after media removal.

Figure 1. CHO M1 response to carbachol.

Table 1. HEK 293 and Jurkat M1 Response to Carbachol (20 nM).

HEK 293 Mean ΔF max (RFU) SD %CV Z′-factor
Fluo-4 NW 37,138 2,551 6.9 0.827
Fluo-4 wash 47,071 3,320 7.1 0.801
Calcium 3 31,800 2,728 8.6 0.779
Jurkat Mean ΔF max (RFU) SD %CV Z′-factor
Fluo-4 NW 6,832 272 3.98 0.842
Calcium 3 5,731 541 9.44 0.694

CHO M1, HEK293 M1 and Jurkat M1 Response to Carbachol.  CHO M1 Cells were treated with 20 nM Carbachol agonist (Figure 1), then assayed using the listed Calcium Assays (results are baseline subtracted and the average of four replicates).  The data in the table was obtained using HEK 293 and Jurkat M1 cells, and are the average of 16 and 8 replicates, respectively.  Z′- factors were calculated using the formula: Z′= 1-3 (SD max + SD min)/(ΔF max- ΔF min), where ΔF max is the average max-min difference upon addition of a maximal concentration of Carbachol agonist, and ΔF min is the average value using only buffer addition.

Figure 2. Fluo-4 NW Assay shows consistent pharmacology for known agonists and inhibitors. CHO M1 Dose Response Curves. Cells were stimulated with Carbachol agonist (Panel A) or Pirenzepine inhibitor (Panel B) over the indicated concentration ranges. Relative Fluorescence (ΔF) values were determined with the listed Calcium Assays according to their respective protocol. The data shows similar pharmacology (EC 50) in each of the various assay methods.


1.    Biotechniques, 34, 164 (2003)
2.    Cell Calcium 11, 57 (1990)


Figure 3.  Pseudocolored images of changes in intracellular free Ca2+ in AtT-20/D16v-F2 cells, monitored at 9-second intervals with fluo-4, AM (F14201, F14202, F14217, F23917). 
Pseudocolored images of changes in intracellular free Ca2+ in AtT-20/D16v-F2 cells, monitored at 9-second intervals with fluo-4, AM (F14201, F14202, F14217, F23917). In order to induce an influx of Ca2+, the cells were depolarized with 50 mM KCl in frame 2 and exposed to 5 µM ionomycin (I24222, a Ca2+ ionophore) in frame 8. The images are pseudocolored according to fluorescence intensity, with red representing high Ca2+ concentrations and blue representing low Ca2+ concentrations. The images were acquired using a fluorescence microscope equipped with a longpass filter set appropriate for fluorescein and a Photometrics Quantix cooled CCD camera.