Calcium Indicators

Calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca²⁺.

Ca²⁺- sensitive fluorescent indicators

Extensive research, including seminal contributions by one of ION’s principal scientists, Dr. Akwasi Minta, has afforded a host of excellent, cell-compatible Ca²⁺ sensors. These molecules are small, synthetic fluorochromes which incorporate a Ca²⁺-binding moiety with excellent selectivity for Ca²⁺ over other physiologically relevant divalent cations, e.g. Mg²⁺. Under conditions where Ca²⁺ is not bound, the fluorescence of the sensor is significantly quenched. When Ca²⁺ is bound, the quenching is relieved, and the fluorescence of the sensor dramatically increases. For some sensors, binding Ca²⁺ also results in a change in the spectral properties of the indicator. For instance, in the case of Fura-2 (below), binding of Ca²⁺ results in a shift of the indicator’s excitation spectrum from a peak at 380 nm in the Ca²⁺ free form to 340 nm in the Ca²⁺-bound form. These indicators are often used in “ratiometric” mode where the Ca²⁺ binding-dependent changes in spectral properties provide a means to control things like photo-bleaching of the indicator and variable optical path length through samples.

Calcium-sensitive fluorescent indicators are compatible with a wide variety of detectors including fluorescent microscopes, plate readers, flow cytometers, and fluorescent indicator-doped solid-state sensors. Over the last 30 years, Ca²⁺-sensitive indicators have found numerous uses ranging from high-spatial resolution imaging of synaptic activity to high-throughput measurements of G protein-coupled receptor (GPCR) activation-evoked increases in intracellular Ca²⁺ for drug discovery. 

Selecting a Ca²⁺ – sensitive fluorescent indicator

Choosing the correct indicator is crucial to the success of an experiment. A number of factors play a role in the decision including the indicator’s wavelength, its Ca²⁺ affinity as well as its cellular loading, retention and localization. ION provides a variety of the most popular and useful fluorescent Ca²⁺ sensors and reagents to help facilitate and empower your research.

 

HeLa cells loaded with Fluo-4 in the presence (+) or the absence (-) of 500 nM of the Ca²⁺  ionophore ionomycin.

Single wavelength indicators

These are the most popular forms of fluorescent Ca²⁺-sensitive indicators. They possess a range of Ca²⁺ affinities and the broadest instrument compatibility. We offer indicators with spectral properties well matched to common fluorescence microscopes, plate readers and flow cytometers using the most widely available FITC-compatible optics (Fluo-2, Fluo-3 and Fluo-4) while excitation sources and filters for Texas Red are compatible with ICR-1.  

 

(EX 490 nm/Em 515 nm, Kd 355 nM) is the most used and the most trusted of the single-wavelength Ca²⁺ indicators. It has been the indicator of choice for countless basic research studies and has been used to screen many tens-of-millions of compounds using high-throughput screening (HTS) to discover pharmacological tools and drugs for GPCRs, and a wide variety of Ca²⁺ permeable ion channels and transporters.

(EX 505 nm/Em 525 nm, Kd 390 nM) is a structurally related predecessor of Fluo-4 which has slightly lower affinity for Ca²⁺ and a slightly red-shifted excitation and emission spectrum. For nearly 10 years it was the most commonly used single wavelength, Ca²⁺ sensor. In most cell types Fluo-4 and Fluo-2 show superior loading compared to Fluo-3, however, some have reported that Fluo-3 is superior in certain instances.

(EX 490 nm/Em 515 nm, Kd 290 nM) Also known as Fluo-2 HA, Fluo-2 offers the same spectral properties as Fluo-4 combined with superior loading in many cell types as well as a slightly higher Ca²⁺ affinity. The improved loading and increased affinity can result in superior sensitivity. 

(EX 580 nm/Em 660 nm, Kd 480 nM) represents a new generation of long sought after fluorescent Ca²⁺ indicators that emit in the red portion of the spectrum. Because of ICR-1’s unique true-red spectral properties, it provides deeper tissue penetration and dramatic background reduction in tissues that have high autofluorescence, in the presence of serum or fluorescent compounds. ICR-1 is also particularly well-suited for use with the most common fluorescent proteins (e.g. GFP/YFP) as well as many optogenetic tools. Unlike some previous red-emitting Ca²⁺-sensitive indicators (e.g. Rhod-2) that concentrate in the mitochondria, ICR-1 enjoys a predominantly cytoplasmic localization similar to the Fluo family of indicators, making it ideal for most intracellular Ca²⁺ measurements.

Dual wavelength indicators

These indicators are particularly useful when more accurate qualification of Ca²⁺ concentrations is desired. However, to utilize the benefits of ratiometric imaging, an instrument (e.g. fluorescence microscope) capable of rapid wavelength switching is required. In the case of Fura-2 (below) an excitation source and optics compatible with 340 nm and 380 nm illumination and ultraviolet (UV) light-compatible sample containers (e.g. microscope slides) are required.

 

(Ex 340/380 nm/505 nm, Kd 145 nM) is by far the mostly commonly used dual-wavelength excitation Ca²⁺-sensitive fluorescent indicator. While Fura-2’s affinity for Ca²⁺ is higher than any of the single wavelength indicators described above, it is still well-suited to measuring changes in cytoplasmic Ca²⁺ under most conditions while enjoying the benefits of ratiometry. In addition to traditional Fura-2, we also offer Fura-2 LR. Fura-2 LR is better retained in some cell lines compared to Fura-2 and thus, may provide superior results.

Indicator forms and formats 

We provide our Ca²⁺-fluorescent dyes in a number of different forms and formats to best suit the intended application including membrane permeable forms, membrane impermeable forms and indicators packaged in kits together with other key reagents optimized to ensure best performance.

Cell permeable, acetoxymethyl group (AM) esters

When first introduced, loading the highly negatively charged Ca²⁺-sensitive fluorescent indicators was very challenging as they existed only as highly negatively charged, membrane impermeable salts. However, the development of forms of these molecules where the negative charges are masked by non-polar esters-linked moieties (AM esters) allow the molecules to enter cells through passive diffusion. An additional advantage of the AM form is that they are also non-fluorescent and non-Ca²⁺ binding. Once inside the cell, ubiquitous intracellular esterase enzymes promote rapid hydrolysis of the AM esters leaving the active, highly polar form of the indicator trapped inside the cell. 

Reagents for improving indicator loading, retention and background suppression

Although the AM forms of ION’s fluorescent Ca²⁺ indicators readily cross the plasma membrane, there are other factors that can affect how well the indicators load into cells. Because AM forms of the indicators are quite hydrophobic, it is common to use the non-ionic surfactant, Pluronic F-127, to improve their solubility in aqueous solution thus improving their loading into cells. Pluronic F-127 at the concentrations employed for loading indicators into cells is generally well tolerated. While Pluronic F-127 isn’t absolutely essential for loading most fluorescent Ca²⁺ indicators into cells, it is highly recommended. 

Another factor that can affect cell loading and intracellular localization is the presence of transport proteins capable of transporting many ion-sensitive, fluorescent indicators. In many cell types, the activity of these transporters can result in compartmentalization of the indicators into intracellular compartments as well as extrusion of the indicators outside of the cell. Both of these processes can dramatically reduce the signal-to-background levels and in some cell types (e.g. CHO cells),  make adequate loading of Ca²⁺-selective fluorescent indicators nearly impossible. Fortunately, inhibitors of these transport proteins can be an effective means of improving cytoplasmic localization and indicator retention. ION offers a convenient, 100X ION-Pro inhibitor solution that may be used in conjunction with any of our Ca²⁺-sensitive fluorescent indicators.

Calcium indicator kits and complete assay solutions

While fluorescent Ca²⁺-sensitive indicators have been in widespread use for decades, not every investigator is familiar with the most up-to-date protocols and additional reagents that may be necessary for their effective use. Therefore, ION provides customers with the option to purchase all reagents commonly used to load our Ca²⁺-sensitive fluorescent indicators into most cell types with clear, easy-to-follow instructions to facilitate successful use of fluorescent, Ca²⁺ sensitive indicators in one convenient package. These Brilliant kits are supplied in a variety of sizes ranging from trial packs to the largest-scale HTS (inquire).

Be Brilliant! Explore our line of Calcium Indicators

Our collection of membrane permeable and impermeable ion indicators targeting Ca²⁺.

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Non-fluorescent calcium (Ca²⁺) chelator, membrane permeable

250
DMSO
$95.00

High-throughput, no wash calcium (Ca²⁺) assay. An optimal solution for measuring intracellular Ca²⁺ dynamics, including effectors such as GPCRs and protein transporters. 100 plates

490/515
FITC, GFP
$2,790.00

High-throughput, no wash calcium (Ca²⁺) assay. An optimal solution for measuring intracellular Ca²⁺ dynamics, including effectors such as GPCRs and protein transporters. 10 plates

490/515
FITC, GFP
$362.00

Green fluorescent, live cell indicator.

495/515
DMSO
FITC
$99.00-$150.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane permeable. Higher Ca²⁺ affinity than Fluo-4.

490/515
290
DMSO
FITC, GFP
$125.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane impermeable. Higher Ca²⁺ affinity than Fluo-4

490/515
290
H2O
FITC
$130.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane permeable

505/525
390
DMSO
FITC
$150.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane impermeable

505/525
390
H2O
FITC
$140.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane permeable

490/515
355
DMSO
FITC, GFP
$140.00

Green fluorescent, calcium (Ca²⁺) indicator, membrane impermeable

490/515
355
H2O
FITC
$140.00

UV-excitable, ratiometric green calcium (Ca²⁺) indicator, membrane permeable

340,380/505
145
DMSO
Fura-2
$95.00

Ratiometric calcium (Ca²⁺) indicator, membrane impermeable

340,380/505
145
H2O
Fura-2
$95.00

Leakage-resistant, ratiometric calcium (Ca²⁺) indicator, membrane permeable

340,380/505
145
DMSO
Fura-2
$150.00

Leakage-resistant, ratiometric calcium (Ca²⁺) indicator, membrane impermeable

340,380/505
145
H2O
Fura-2
$150.00

Red fluorescent, calcium (Ca²⁺) indicator, membrane permeable. Optimal for multiplexing and tissue imaging. Fluorescence lifetime and multiphoton compatible.

580/660
480
DMSO
Texas Red
$455.00

Red fluorescent, calcium (Ca²⁺) indicator, membrane impermeable. Optimal for multiplexing and tissue imaging. Fluorescence lifetime and multiphoton compatible.

580/660
480
H2O
Texas Red
$455.00
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