Calcium Indicators

To study the roles of the important intracellular messenger calcium in cells, often it is essential to quantitatively monitor its concentration. The most widely used method of Ca2+ detection is by the use of fluorescent Ca2+ indicators, a technique pioneered by Roger Tsien and colleagues. Ca2+ indicators are fluorophores coupled to a BAPTA calcium chelator structure. Binding of calcium to the chelator portion of the molecule causes a spectral shift in the dye fluorescence and/or increased dye fluorescence. A variety of indicators are available that vary in their Ca2+ dissociation constants (Kd) or Ca2+ response range, excitation/emission wavelengths, spectral shift, and relative fluorescent quantum yields.

You should select a Ca2+ indicator that best suits your needs in consideration of the biological system, instrument settings, and other fluorescent probes used in the experiment. The indicator Kd values give an estimate of the Ca2+ concentration response, usually 0.1 Kd to 10 Kd, with the caveat that Kd values measured in vitro may differ considerably from those in cells due to differences in ionic strength, pH, viscosity, and Ca2+ buffering by lipids and proteins.

Ratiometric vs. Non-Ratiometric Indicators

Upon binding to calcium, ratiometric indicators undergo a shift either excitation wavelength, emission wavelength, or both. Ca2+concentration is determined by measuring indicator fluorescence at two different excitation and emission settings (corresponding to the maxima with no Ca2+and high Ca2+) and determining their ratio. These indicators have the advantage of being internally controlled, and therefore avoid artifacts from uneven dye loading or photobleaching between samples. Ratiometric measurements may require specialized imaging setups or filter cubes. Non-ratiometric indicators show increased fluorescence intensity upon binding to calcium, with no shift in excitation/emission wavelengths.

Cell Membrane-Permeant AM Esters

In their salt forms, indicators are water-soluble and membrane impermeant, so loading them into cells requires microinjection. AM esters are membrane-permeant forms of indicators that themselves do not bind ions. However, once they have entered cells, they are hydrolyzed by esterases to release the parent ion indicator. Thus, cells or tissues can be loaded with indicator by simply incubating them with a buffer containing the AM ester form. Biotium also supplies Pluronic® F-127, a mild non-ionic detergent that can facilitate cell loading of AM ester compounds, and high grade anhydrous DMSO for preparing AM Ester stock solutions.

Properties of Calcium Indicators

Indicator	MW1	Ratiometric?	Excitation2	Emission2	Kd3	Membrane-Impermeant Salts	Membrane-Permeant AM Esters
Fluo-3	770	No	506 nm	525 nm	390 nM	Fluo-3, Pentaammonium Fluo-3, Pentapotassium Fluo-3, Pentasodium	Fluo-3, AM Ester Fluo-3 AM Ester, 1 mM in DMSO
Fluo-4	737	No	494 nm	506 nm	335 nM	Fluo-4, Pentapotassium	Fluo-4, AM Ester
Mag-Fluo-4	682	No	493 nm	517 nm	22 uM	Mag-Fluo-4, Tetrapotassium	Mag-Fluo-4, AM Ester
Fura-2	642	Yes	363/335 nm	512/505 nm	145 nM	Fura-2, Pentaammonium Fura-2, Pentapotassium Fura-2, Pentasodium	Fura-2, AM Ester Fura-2, AM Ester, 1 mM in DMSO
Indo-1	650	Yes	349/331 nm	482/398 nm	230 nM	Indo-1, Pentaammonium Indo-1, Pentapotassium Indo-1, Pentasodium	Indo-1, AM Ester
Furaptra (Mag-Fura-2)	435	Yes	369/330 nm	511 nm	1.9 mM (Mg2+) 25 uM (Ca2+)	Furaptra , Pentapotassium Furaptra, Pentasodium	Furaptra, AM Ester
Rhod-2	755	No	556 nm	576 nm	1 uM	Rhod-2, Triammonium Rhod-2, Tripotassium Rhod-2, Trisodium	Rhod-2, AM Ester
Rhod-590	912	No	595 nm	616 nm	610 nM	Rhod-590, Tripotassium	Rhod-590, AM Ester

1. Free acid form
2. For non-ratiometric indicators, Ex/Em with calcium is shown; for ratiometric indicators, wavelengths with no calcium/high calcium are shown
3. Ca2+ dissociation constant measured at 22°C, pH 7.2 buffer

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