Novel improved Ca2+ indicator dyes on the market-a comparative study of novel Ca2+ indicators with fluo-4

Calcium ions (Ca²⁺) are pervasive second messengers responsible for the regulation of many physiological and biological processes of cells and organisms. In neuromuscular junction signaling pathways, Ca²⁺ promotes the release of the neurotransmitter acetylcholine from motor neurons for the activation of skeletal muscle concerned with locomotion. Calcium acts as a cell signaling molecule monitoring enzymatic activity, ion channel permeability and ion pump activity; and promotes healthy and proper bone formation during human development. Ongoing discoveries of calcium's versatility and the ensuing need to study calcium's involvement in many prominent physiological processes triggered the development of Fluo-3 and Fluo-4 Ca²⁺ indicating probes for the visualization and quantification of calcium transients. Since their discovery in the 1980s, there has been an increase in the research and development of novel Ca²⁺ indicators with improved functionality and sensitivity in correlation to Fluo-3 and Fluo-4. However, Fluo-3 and Fluo-4 continue to dominate the market even with drawbacks, such as high background interference, associated with its employment. To promote awareness of these novel Ca²⁺ indicators, an increasing number of comparative studies have highlighted the enhancements and benefits associated with these newly developed Ca²⁺ indicators, such as Cal-520^®, in comparison to Fluo-3 and Fluo-4

A recent comparative study performed by members of the Department of Life, Health and Chemical Sciences at the Open University in London investigated spectral characteristics from select novel Ca²⁺ indicators with the well-established Fluo-4 (Molecular Probes). Of the novel 'green' Ca²⁺ indicators tested were AAT Bioquest's Cal-520^® (Catalog: 21130) and Enzo Life Science's FluoForte. Subjected to identical test condition, each Ca²⁺ indicator was evaluated on their intracellular dye retention and fluorescent abilities. Dr. Rietdorf and her groups findings revealed and solidified Cal-520^®'s superiority over Fluo-4 and the other commercially available 'green' Ca²⁺ indicators. Cal-520^® boasted the brightest starting fluorescence (25% greater than Fluo-4; Figure 1) and the largest change in fluorescent intensity (delta f/f0; Figure 2). Cal-520^®'s minimal loss of fluorescence during imaging and complete dye retention of Cal-520^® after incubation demonstrates its stability and suitability for long-duration imaging applications (Figure 3 and 4).





Open University's comparative study also investigated two novel 'red' Ca²⁺ indicators, AAT Bioquest's Cal-590™ (Catalog: 20510) and Enzo Life Sciences GFP-certified FluoForte, subjecting both to identical experimental parameters performed previously with the 'green' Ca²⁺ indicators. Their findings coincide with our understanding of fluorescence. Longer excitation wavelengths required for 'red' Ca²⁺ indicators, results in a weaker fluorescence signal, smaller change in fluorescence intensity and narrower dynamic range (Figure 1 - 4). These spectral properties of 'red' Ca²⁺ indicators prove beneficial as replacements for Fluo-4 and calcium indicators experiencing problems regarding signal saturation. The essence of this study alludes to Cal-590™'s superiority over GFP-certified FluoForte as a second dye for multiplexing Ca²⁺ assays. Excitation of both dyes at 488 nm revealed Ca²⁺ sensitive fluorescence by Enzo's GFP-certified FluoForte, which is indicative of background interference making it difficult to use for simultaneous imaging. Contrastingly, Cal-590™ did not show any fluorescence when excited by the 488 nm light, eliminating potential cross-talk during multiplexing assays involving simultaneous excitation with 488 and 560 nm lasers.

Another active area investigating the comparison of fluorescent Ca²⁺ indicators is the detection and observation of localized subcellular changes in Ca²⁺. In this study performed by the University of California, Irvine, fluorescent Ca²⁺ indicators were evaluated on their spectral properties and sensitivity at detecting local Ca²⁺ signals evoked by inositol triphosphate (IP₃). Because Ca²⁺ flux occurs rapidly, sensitive high-speed camera-based fluorescence microscopy was utilized to detect and analyze local, subcellular Ca²⁺ signals. Of the many novel 'green' Ca²⁺ indicators tested were AAT Bioquest's Cal-520^® and Invitrogen's Oregon Green BAPTA-1 (OGB-1) and Fluo-4. Each Ca²⁺ indicator was loaded into human neuroblastoma SH-SY5Y cells using acetoxymethyl (AM) ester forms to facilitate intracellular uptake. Stimulation of localized subcellular Ca²⁺ signals were induced by photolysis flashes that were calibrated to the precise intensity and duration needed to generate a detectable and measurable local response, without exciting a global rise in cytosolic calcium.

Data gathered from this investigation reinforced Cal-520^®'s superiority as a novel 'green' Ca²⁺ indicator. Cal-520^®'s low baseline fluorescence and largest increase in fluorescence upon Ca²⁺ binding is indicative of its large dynamic range.Cal-520^®'s large dynamic range improves assay sensitivity, detecting acute changes in subcellular Ca²⁺ concentrations. In kinetic assays such as monitoring Ca²⁺ flux or the opening/closing of Ca²⁺ channels, Cal-520^®'s large dynamic range is excellent at accommodating the larger fluorescent signal changes that are presumably to occur during these Ca²⁺ kinetic assays. When used in conjunction with fluorometers optimized for superior dynamic range detectability, a more accurate depiction of the analyte concentrations can be obtained. Of all the calcium indicators, Cal-520^® showed optimal kinetic resolution detecting the fastest rise times in signal intensity from 20-80%.

Open University and UCI's comparative studies confirmed Cal-520^®'s superiority as a sensitive and robust calcium indicator. The Cal-500 dye series provides an excellent means for simultaneous Ca²⁺ imaging because Cal-590™ cannot be excited by the 488 nm light. Furthermore, Cal-520^® and Cal-590™'s remarkable leakage resistance maintains optimal intracellular dye retention improves signal-to-noise ratio, dynamic range and allows for the continuous generation of a strong fluorescent signal. The Cal-500 dye series is enhanced for in vivo and in vitro experiments from detecting and monitoring calcium transients to neural imaging.

References

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1. Lock, Jeffrey T., Ian Parker, and Ian F. Smith. "A comparison of fluorescent Ca2+ indicators for imaging local Ca2 signals in cultured cells." Cell Calcium 58.6 (2015): 638-48. Web.
2. Rietdorf, Katja; Chehab, Tala; Allman, Sarah and Bootman, Martin D. (2014). Novel improved Ca²⁺ indicator dyes on the market-a comparative study of novel Ca²⁺ indicators with fluo-4. In: Calcium Signalling: The Next Generation, 9-10 October 2014, London, UK.
3. Cal-520^® AM. AAT Bioquest, n.d. Web. 28 June 2016