Introduction to Ca²⁺ Measurements with Fluorescent Indicators—Section 19.1

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• Selection Criteria for Fluorescent Ca2+ Indicators

Fluorescent probes that show a spectral response upon binding Ca²⁺ have enabled researchers to investigate changes in intracellular free Ca²⁺ concentrations using fluorescence microscopy, flow cytometry and fluorescence spectroscopy. The properties and applications of these fluorescent indicators—most of which are derivatives of the Ca²⁺ chelators EGTA, APTRA and BAPTA —have been extensively reviewed. Several earlier reviews of these ion indicators also contain useful technical information.

We discuss chemical Ca²⁺ indicators according to their excitation requirements in Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2 and Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3, and their high-molecular weight conjugates are described in Fluorescent Ca2+ Indicator Conjugates—Section 19.4. Protein-based Ca²⁺ sensors are discussed in Protein-Based Ca2+ Sensors—Section 19.5.

We offer the widest selection of fluorescent indicators available for detecting changes in intracellular Ca²⁺ over the range of <50 nM to >50 µM (Summary of Molecular Probes fluorescent Ca2+ indicators—Table 19.1). As the primary suppliers of fura-2, indo-1, fluo-3 (), fluo-4 and rhod-2, we also offer many specialized indicators for intracellular Ca²⁺. Our fura-4F, fura-6F and fura-FF indicators provide increased response sensitivity to intracellular Ca²⁺ concentration in the 0.5–5 µM range, as compared with fura-2. The fluo-3, fluo-4, Oregon Green 488 BAPTA, Calcium Green, X-rhod-1 and Fura Red indicators and their variants enable Ca²⁺ detection in confocal microscopy and high-throughput G protein–coupled receptor (GPCR) screening applications. In addition, we offer indicators that are conjugated to high– or low–molecular weight dextrans for improved cellular retention and less compartmentalization (Fluorescent Ca2+ Indicator Conjugates—Section 19.4). We strive to provide the highest-purity indicators available anywhere. The AM ester forms of most of our indicators are typically at least 95% pure by HPLC analysis, although purity often exceeds 98%. Furthermore, the AM esters of many of the Ca²⁺ and Mg²⁺ indicators are available in sets of 50 µg for more convenient handling and reduced risk of deterioration during storage. For high-throughput screening applications, fluo-4 AM is offered in special multi-unit packages (F14202), as well as in application-specific Fluo-4 NW and Fluo-4 Direct Calcium Assay Kits (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3).

A number of factors should be considered when selecting a fluorescent Ca²⁺ indicator, some of which are summarized in Summary of Molecular Probes fluorescent Ca2+ indicators—Table 19.1 and include the following:

• Indicator form (salt, AM ester or dextran conjugate), which influences the cell-loading method and affects the indicator's intracellular distribution and retention (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). The salt and dextran forms are typically loaded by microinjection, microprojectile bombardment or electroporation or by using the Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) (Choosing a Tracer—Section 14.1, Techniques for loading molecules into the cytoplasm—Table 14.1). In contrast, the cell-permeant acetoxymethyl (AM) esters can be passively loaded into cells, where they are cleaved to cell-impermeant products by intracellular esterases.
• Measurement mode, which is dictated by whether qualitative or quantitative ion concentration data are required. Ion indicators that exhibit spectral shifts upon ion binding can be used for ratiometric measurements of Ca²⁺ concentration, which are essentially independent of uneven dye loading, cell thickness, photobleaching effects and dye leakage (Loading and Calibration of Intracellular Ion Indicators—Note 19.1). Excitation and emission wavelength preferences depend on the type of instrumentation being used, as well as on sample autofluorescence and on the presence of other fluorescent or photoactivatable probes in the experiment.
• Dissociation constant (K_d), which must be compatible with the Ca²⁺ concentration range of interest. Indicators have a detectable response in the concentration range from approximately 0.1 × K_d to 10 × K_d. For ratiometric indicators, the Ca²⁺ response range is also somewhat dependent on the measurement wavelengths used. The K_d of Ca²⁺ indicators is dependent on many factors, including pH, temperature, ionic strength, viscosity, protein binding and the presence of Mg²⁺ and other ions. Consequently, K_d values for intracellular indicators are usually significantly higher than corresponding values measured in cell-free solutions (Comparison of in vitro and in situ Kd values for various Ca2+ indicators—Table 19.2).

Intracellular calibration of Ca²⁺ indicators may be achieved either by manipulating Ca²⁺ levels inside cells using an ionophore or by releasing the indicator into the surrounding medium of known Ca²⁺ concentration via detergent lysis of the cells. We also offer several compounds and buffers for measuring and manipulating intracellular and extracellular Ca²⁺. These products, which are discussed in Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8, include caged Ca²⁺ reagents and caged chelators (NP-EGTA, DMNP-EDTA and diazo-2), as well as Calcium Calibration Buffer Kits, BAPTA-derived buffers, ion-selective chelating polymers (Calcium Sponge) and the important Ca²⁺ ionophores ionomycin, A-23187 and its nonfluorescent analog, 4-bromo A-23187. Reagents for probing Ca²⁺ regulation and second messenger activity are described in more detail in Probes for Signal Transduction—Chapter 17. Our reagents for the study of Ca²⁺ channels are described in Probes for Ion Channels and Carriers—Section 16.3.