Fluorescent Calcium Indicators & Related Reagents
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|
|Bis-Fura-2||779||Yes||363/335 nm||512/505 nm||370 nM||Bis-Fura-2, Hexapotassium||—|
|Fluo-3||770||No||506 nm||525 nm||390 nM||Fluo-3, Pentaammonium|
|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|
|Fura-2||642||Yes||363/335 nm||512/505 nm||145 nM||Fura-2, Pentaammonium|
|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, AM Ester|
|Furaptra (Mag-Fura-2)||435||Yes||369/330 nm||511 nm||1.9 mM (Mg2+)|
25 uM (Ca2+)
|Furaptra , Pentapotassium|
|Furaptra, AM Ester|
|Rhod-2||755||No||556 nm||576 nm||1 uM||Rhod-2, Triammonium|
|Rhod-2, AM Ester|
|Rhod-590||912||No||595 nm||616 nm||610 nM||Rhod-590, Tripotassium||Rhod-590, AM Ester|
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
Calcium Chelators & Accessory Reagents
BAPTA and its derivatives are calcium chelators that are commonly used to generate calcium buffers with well-defined calcium concentrations. By introducing the chelators into cells by microinjection or with AM ester forms of the chelators, one can control the cytosolic calcium concentration, an important means to study the roles of calcium.
Key advantages of these calcium chelators include relative insensitivity toward intracellular pH change and fast release of calcium. Biotium offers several BAPTA chelators with calcium dissociation constants covering the biologically significant range from 10-7 to 10-2 M.
Properties of BAPTA Chelators
|Chelator||MW1||Kd2||Membrane-Impermeant Salts||Membrane-Permeant AM Esters|
|BAPTA||477||No Mg2+: 0.59 mM|
1 mM Mg2+: 0.70 mM
|BAPTA, AM Ester|
|5,5′-Dibromo BAPTA||635||No Mg2+: 3.6 mM||5,5′-Dibromo BAPTA, Tetrapotassium||—|
|5,5′-Difluoro BAPTA||513||No Mg2+: 0.61 mM|
1 mM Mg2+: 0.72 mM
|5,5′-Difluoro BAPTA, Tetrapotassium||5,5′-Difluoro BAPTA, AM Ester|
|5,5′-Dimethyl BAPTA||505||No Mg2+: 0.16 mM|
1 mM Mg2+: 0.44 mM
|5,5′-Dimethyl BAPTA, Tetrapotassium||5,5′-Dimethyl BAPTA, AM Ester|
|5-Methyl-5′-nitro BAPTA||536||No Mg2+: 0.53 mM||5-Methyl-5′-nitro BAPTA, Tetrapotassium||—|
|5-Mononitro BAPTA||522||No Mg2+: 0.94 mM||—||—|
|4-Trifluoromethyl BAPTA||545||No Mg2+: 0.57 mM||—||—|
2 Cell Calcium 10, 491 (1989).
Calcium Ionophores and Caged Calcium
Calcium ionophores are ion carriers that facilitate the equilibration of calcium across cellular membranes. They are useful tools for calibrating calcium indicators and manipulating intracellular calcium concentration.
A-23187 (calcimycin, calcium ionophore III) rapidly equilibrates intracellular and extracellular calcium concentrations. It is commonly used for in situ calibration of fluorescent calcium indicators. The ionophore also allows Mn<sup>2+</sup> to enter the cells and quench intracellular fluorescence of calcium indicators. A-23187 has blue fluorescence, which can contribute to fluorescence background when used with UV-excited calcium indicators. 4-Bromo A-23187 is non-fluorescent ionophore, preferred for calibrating UV-excited Ca<sup>2+</sup> indicators such as Indo-1 and Fura-2.
Ionomycin is commonly used to modify intracellular calcium concentrations and to calibrate fluorescent calcium indicators. It also is used to stimulate cytokine production and cell proliferation in lymphocytes. In other cell types it can induce apoptosis and inhibit proliferation.
DMNP-EDTA (also known as DM-Nitrophen™) is a caged Ca2+ chelator. Exposure to UV light cleaves the chelator, allowing controlled release of calcium. Upon photolysis, the Kd for Ca2+ increases from 5 nM to 3 mM, resulting in a pulse of free Ca2+.
Calcium Calibration Buffers
The Calcium Calibration Buffer Kit is used to prepare buffers with a range of accurate calcium concentrations, and is useful for the calibration of fluorescent calcium indicators. The kit contains 50 mL each of zero calcium buffer and 40 uM free Ca2+ buffer, with a detailed protocol for combining the two buffers to obtain calibration curve from 0.017-39.8 uM free calcium.
TPEN is an excellent heavy metal chelator that does not affect calcium, magnesium, sodium, or potassium concentrations. Therefore it is useful tool for distinguishing the effects of heavy metals like zinc, iron, copper, and manganese on fluorescent indicators for calcium, magnesium, sodium and potassium.
Accessory Reagents for Indicator Dyes
Pluronic® F-127 is a mild detergent useful for solubilizing AM esters for cell loading. We also offer anhydrous DMSO in convenient 10 mL size, recommended for preparing stock solutions of AM esters, which are susceptible to hydrolysis. EDC (or EDAC) has been found to be useful for fixing chelators in situ, for subsequent fixation and staining.
Accessories for Calcium Indicators & Other Ion Indicators
|Calcium Calibration Buffer Kit (10 mM CaEGTA buffer and 0 mM CaEGTA buffer)||59100||50 mL each buffer|
|EDC (EDAC)||59002||100 mg|
|Pluronic F-127||59000||1 g|
|Pluronic F-127, 20% in DMSO||59004||1 mL|
|Pluronic F-127, 10% in sterile dH2O||59005||30 mL|
|DMSO, Anhydrous||90082||10 mL|
Other Ion Indicators
The fluorescence of SPQ 6(-methoxy-N-(3-sulfopropyl)quinolinium) is specifically quenched by chloride via collision. Therefore, chloride concentration is measured by monitoring the degree of fluorescence decrease. The dye can be loaded into cells by hypotonic shock. The dye has excitation/emission at 344/443 nm.
MQAE (N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide) is an improved chloride indicator that has greater sensitivity to chloride (Ksv= 200 M-1) than SPQ (Ksv= 118 M-1) and higher fluorescence quantum yield. The ester group of MQAE may slowly hydrolyze inside cells, resulting in a change in its fluorescence response. The dye has excitation/emission at 350/460 nm.
Zinc is believed to be involved in the suppression of apoptosis and play important roles in many neural activities. Zinquin is an UV-excitable, blue fluorescent zinc indicator with excitation/emission at 350/460 nm. Zinquin free acid is membrane-impermeant, while Zinquin ethyl ester is membrane-permeable and is hydrolyzed into Zinquin free acid after entering cells.
Chloride & Zinc Indicators
|Product||Indicator||Membrane permeability||Ex/Em||Catalog number||Unit size|
|SPQ||Chloride||Impermeant||344/443 nm||52010||50 mg|
|MQAE||Impermeant||350/460 nm||52011||100 mg|
|Zinquin free acid||Zinc||Impermeant||350/460 nm||52022||5 mg|
|Zinquin ethyl ester||Permeant||350/460 nm||52020||5 mg|
BCECF pH Indicators
BCECF is the most widely used fluorescent pH sensor. With a pKa of 6.97, close to physiological pH, it can detect changes in cytosolic pH with high sensitivity. At low pH, the dye is weakly fluorescent but becomes more fluorescent with increasing pH. The excitation spectrum of the dye undergoes a slight shift with pH change, while the wavelength of the emission maximum remains unchanged. The pH is determined ratiometrically by the relative fluorescent intensities at 535 nm when the dye is excited at 439 nm and 505 nm. Available in membrane-impermeant free acid and cell-permeable AM ester forms.
Flubida & Flubi-2 pH Indicators
Flubida-2 has been used to detect pH at a specific site in a cell such as cell organelles by directing the probe to where avidin-chimera proteins are located. The probe is a conjugate of biotin and fluorescein diacetate, which is nonfluorescent until the probe has entered the cells and is hydrolyzed by cellular esterases. Flubida-2 is membrane-permeable and can be delivered into cells via simple incubation. Flubi-2 is the membrane-impermeable hydrolyzed product of Flubida-2. Its spectral properties and pH response are similar to those of BCECF.
Carboxyfluorescein pH Indicators
Carboxyfluorescein (FAM) has a pKa of 6.5 and can be used as a pH indicator or cellular tracer. Its excitation spectrum and fluorescence response to pH are similar to those of BCECF. Carboxyfluorescein is also available in membrane permeable diacetate form (CFDA). CFDA-SE is a membrane-permeable amine-reactive form that covalently reacts with cytoplasmic proteins for stable labeling.
5-(and-6)-Carboxy-2′,7′-dichlorofluorescein is similar to carboxyfluorescein, but has a pKa of 4.8, and therefore is useful for detecting pH in more acidic environment. It is also available in membrane-permeable diacetate, and membrane-permeable, amine-reactive diacetate succinimidyl ester forms.
The pKa of 5-carboxy-2’,7’-dichlorosulfonefluorescein is near 4.0 and thus the dye is potentially an excellent pH indicator for acidic organelles.
Fluorescein derivatives are available as single isomer or mixed isomers; the isomers have the same spectral properties. For certain coupling reactions, single isomer may be preferable, but mixed isomers are suitable for most applications.
Membrane-Permeant pH Indicators
|Product||pKa||Catalog number||Unit size|
|BCECF AM Ester, 1 mg/mL in DMSO||pKa 6.97||51009||1 mL|
|BCECF AM Ester||51011|
|10 x 100 ug
20 x 50 ug
|5-Carboxyfluorescein diacetate, single isomer (5-CFDA)||pKa 6.5||51018||100 mg|
|6-Carboxyfluorescein diacetate, single isomer (6-CFDA)||51021||100 mg|
|5-(and 6)-Carboxyfluorescein diacetate, mixed isomers (5(6)-CFDA)||51014||100 mg|
|5-(and-6)-Carboxyfluorescein diacetate, succinimidyl ester (CFDA, SE)||90041||25 mg|
|5-(and-6)-Carboxy-2′,7′-dichlorofluorescein diacetate||pKa 4.8||51016||100 mg|
|5-(and-6)-Carboxy-2′,7′-dichlorofluorescein diacetate, succinimidyl ester (SE)||90040||25 mg|
Membrane-Impermeant pH Indicators
|Product||pKa||Catalog number||Unit size|
|BCECF, free acid||pKa 6.97||51010||1 mg|
|5-Carboxyfluorescein (5-FAM, single isomer)||pKa 6.5||51019||100 mg|
|6-Carboxyfluorescein (6-FAM, single isomer)||51020||100 mg|
(5(6)-FAM, mixed isomers)
|5-Carboxy-2′,7′-dichlorofluorescein||pKa 4.8||51017||100 mg|
|5-Carboxy-2′,7′-dichlorosulfonefluorescein||pKa 4||51023||10 mg|
Membrane Potential Dyes
Slow-Responding Membrane Potential Dyes
Translational (or slow-responding) membrane potential dyes undergo a change in their membrane distribution as a result of changes in membrane potential.
The fluorescence of DiBAC4(3) is enhanced with membrane depolarization. The rate of fluorescence response of the dye is slower than styryl dyes like the ANEPPS dyes (see below), but the fluorescence change is significantly larger.
DiOC2(3) has been used for measuring membrane potential in bacteria. The green fluorescent dye forms red fluorescent aggregates with increasing membrane potential, allowing ratiometric potential measurements.
DiOC5(3) and DiOC6(3) are two of the most widely used carbocyanine dyes for membrane potential measurements.
Tetramethylrhodamine ethyl ester (TMRE) and Tetramethylrhodamine methyl ester (TMRM) can be used for quantitative measurements of membrane potential and mitochondrial membrane potential.
DiO/DPA Membrane Potential Kit
The membrane localization of the fluorescence quencher dipicrylamine (DPA) is a function of the polarity and magnitude of membrane potential. The DiO/DPA system detects cytoplasmic membrane potential changes using the principle of fluorescence resonance energy transfer (FRET). The green fluorescent membrane dye DiO is a “stationary” FRET donor while DPA acts as a mobile FRET acceptor, resulting in a membrane potential-dependent quenching of fluorescence by FRET. The DiO/DPA system has been reported to produce a fluorescence signal change of >56% in HEK-293 cells and >25% in neuronal cultures and brain slices per 100 mV membrane potential change.
Fast-Responding Membrane Potential Dyes
Fast-responding membrane potential dyes are styryl dyes that undergo changes in fluorescence intensity in response to changes in membrane potential, on the order of 2-10% change in fluorescence per 100 mV. The dyes also undergo spectral shift with changes in membrane potential, allowing ratiometric measurements. Fast response dyes have been used to measure electrical activity in neural and cardiac cells.
Di-4-ANNEPS has been used for studies of human stem cell-derived cardiomyocytes. Di-8-ANNEPPS is more hydrophobic and better retained in the outer leaflet of the plasma membrane than Di-4-ANNEPS, and therefore is more suitable for long-term membrane potential studies. It is also more photostable and less phototoxic than Di-4-ANNEPS.
Di-2-ANEPEQ (also known as JPW 1114) is a highly water soluble fast-responding dye that is usually introduced into cells by microinjection. Di-8-ANEPQ and Di-12-ANEPQ are successively more hydrophobic, and have been used for potential-sensitive retrograde labeling of neurons.
RH237, RH414, RH421, and RH795 are fast-responding potentiometric probes generally used for functional imaging of neurons. RH421 exhibits >20% fluorescence change per 100 mV on neuroblastoma cells. These dyes can differ in their physiological effects, for example RH414 causes arterial constriction during cortex staining, while the spectrally similar dye RH795 does not.
Fast-Responding Membrane Potential Dyes
|Di-2-ANEPEQ (JPW 1114)||See Note 2||61013|
|DiO/DPA Membrane Potential Kit||484/501 nm||30037|
2Spectrally similar to the ANEPPS dyes.