Fluo-4 NW (no-wash) calcium assay kit provides a proprietary calcium assay formula that requires neither Washing steps also do not require quenching group dyes. The fluo-4 NW Assay Kit achieves higher fluorescence intensities compared to standard fluo-3 and fluo-4 assay kits that include a wash step. Eliminating the wash step reduces variability and improves Z´ values compared to the standard fluo-4 assay kit, while enabling a simpler, faster assay.

fluo-4NW indicator is non-fluorescent and stable for several hours in buffers with a pH of 7–7.5 and therefore spontaneously converts to a Ca²⁺-sensitive form It is not the main cause of background fluorescence. Removing the medium before adding the indicator dye to the well eliminates the contribution of the growth medium (e.g., esterase activity, proteins that interact with the receptor of interest, or phenol red) to the baseline fluorescence.

Another reason for the formation of extracellular potential fluorescence is that the indicator is excreted out of the cell by organic anion transporters. Probenecid is commonly used to inhibit this transport and reduce baseline signal. We have synthesized a proprietary water-soluble probenecid that is provided with the Fluo-4 NW Calcium Assay Kit. The advantage of this form of probenecid is that it dissolves easily in buffers and is safer to use than the free acid, which requires caustic 1 M sodium hydroxide to dissolve. The Fluo-4 NW Calcium Assay Kit is designed for use in microplates and HTS and can be used to measure both adherent and non-adherent cells.

Fluo-4 AM is a fluorescent Ca²⁺ indicator widely used in high-throughput screening (HTS) applications to detect agonist-stimulated and antagonist-inhibited calcium. Signal transduction was measured intracellularly. With the advantages of visible wavelength excitation (compatible with argon ion laser sources), high sensitivity, and significantly enhanced fluorescence upon binding to Ca²⁺, it has become a tool for characterizing the pharmacology and function of G protein-coupled receptors (GPCRs). Indicator of choice. Because of these properties, fluo-4 AM is attractive not only for microplate screening applications but also for microscopy and flow cytometric analysis.