The Fluo-4 NW ( No Wash) Calcium Assay is the next generation calcium indicator from the industry leading Fluo calcium indicator product family. Developed specifically to meet the requirements of automated screening (HTS) applications, the Fluo-4 NW Assay provides superior performance without a quencher dye, combined with the convenience of a no-wash format. This new approach to calcium detection minimizes the potential of non-specific assay interference and reduces %CVs, thereby improving data quality using adherent and suspension cell formats.

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The Advantages of the Fluo-4 NW Assay:

• Measure cell signaling events in a biological context by eliminating potential non-specific interactions that may be caused by addition of a quenching dye
• Achieve higher Z-factors by reducing the variability associated with wash steps
• Robustly screen both adherent & non-adherent cell lines in high throughput mode
• Multiplex using GeneBLAzer® technology to provide more content and validate hits

Better Signal, Better Performance

Homogeneous fluo-4 assays typically incorporate a quencher dye or a washing step in order to minimize extracellular fluorescence (background). However, both quenching dyes and wash steps can have adverse effects on the biological system being analyzed, potentially resulting in an inaccurate measurement. Quenchers have been shown to interfere with some receptor systems of interest¹, and wash steps introduce an extra source of variability (potentially resulting in higher %CVs) and increase the risk of losing non-adherent cells. The new Fluo-4 NW Assay has a unique formulation that eliminates the need for either a quencher dye or a wash step, while delivering a larger increase in fluorescence intensity than either the standard fluo-4 AM assay with a wash step or the Calcium 3 Assay (Molecular Devices). This new formulation results in a more reliable Ca2+ assay designed to meet the requirements of automated applications.

The Flexibility of a Homogeneous Assay

The fluo-4 NW assay protocols are written to streamline assays using both adherent and suspension cell formats. For adherent mammalian cells in microplates, remove the growth medium, incubate the cells in dye-loading solution for up to one hour, stimulate the cells with agonist and measure fluorescence in a microplate fluorometer. For non-adherent cells, we recommend centrifuging the cells, resuspending and dispensing them into a microplate prior to dye loading.

A Safer and Easier to Use Probenecid

Probenecid is widely used too block efflux of intracellular dyes and was first described by Virgilio². The commonly used free acid form of probenecid is often difficult to dissolve, requiring 1 M NaOH to get it into solution. Our new water-soluble probenecid is dissolves quickly in assay buffer, so it’s ready to use immediately. Moreover, it is more effective than the acid form on an equimolar basis, likely due to its better solubility. Water-soluble probenecid included in the Fluo-4NW Calcium Assay Kits and is also available separately.

Product Images:

Figure 1.

Tables 1 & 2. HEK 293 and Jurkat M1 Response to Carbachol(20nM)

CHO M1, HEK293 M1 and Jurkat M1 Response to Carbachol. CHO M1 Cells were treated with 20 nM Carbachol agonist (top panel), then assayed using the listed Calcium Assays (results are baseline subtracted and the average of four replicates). The data in the tables were 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. (click image for larger view)

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)