Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate reading. Most of these fluorescent indicators are derivatives of BAPTA chelators that incorporate a PET system responsive to calcium. FLIPR® and FlexStation™ instruments of Molecular Devices Corp. have enabled high throughput measurement of calcium for GPCR and ion channel research.
There are quite a few factors that need be considered when selecting a fluorescent Ca2+ indicator. These include:

• Spectral Properties: For UV excitation, Indo-1 and Fura-2 are widely used. Fluo-3 is preferred for 488 excitation while Rhod-2 and X-rhod are used for red emissions.
• Measurement Mode: 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. 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. Indo-1 and Fura-2 are primary choice for ratiometric measurements while Fluo-3 and Rhod-2 are predominantly used for single wavelength measurements.
• Permeability of Indicator (salt or AM ester): The salt forms are typically loaded into cells by microinjection, microprojectile bombardment or electroporation, or used for extracellular assays. 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.
• Dissociation Constant (Kd): The desired indicators must have a proper Kd compatible with the Ca2+ concentration range of interest. Indicators have a detectable response in the concentration range from approximately 0.1Kd to 10Kd. The Kd values of Ca2+ indicators are dependent on many factors, including pH, temperature, ionic strength, viscosity, protein binding and the presence of Mg2+ and other ions. Consequently, Kd values for intracellular indicators are usually significantly higher than corresponding values measured in cell-free solutions.

Whatever the specific size, vialing, purity that your assay requires, contact contact Nathalie BERVAS, Brand Manager (nathalie.bervas@tebu-bio.com) if you don't find from the list the exact reagent you need!