Calcium indicators, molecules that exhibit an increase in fluorescence upon binding Ca²⁺, can be used to measure the activity of calcium channels, GPCRs and more.

Calcium Assays and Indicators

Calcium Assays

These assays are compatible with a wide variety of detectors including fluorescent microscopes, high-throughput screening compatible plate readers (FLIPR), flow cytometers, and more. Over the last 30 years, fluorescence-based calcium mobilization assays have revolutionized cell-based research to shed light on signaling pathways, discover new drugs, and better understand cardiomyocyte function.

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, 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 assays have found numerous uses ranging from high-spatial resolution imaging of synaptic activity in neurons and brain tissue, to high-throughput measurements of G protein-coupled receptor (GPCR) activation-evoked increases in intracellular Ca²⁺ for drug discovery.

Calcium assays have found numerous uses ranging from high-spatial resolution imaging of synaptic activity in neurons and brain tissue, to high-throughput measurements of G protein-coupled receptor (GPCR) activation-evoked increases in intracellular Ca²⁺ for drug discovery.

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

Selecting a fluorescent calcium assay

Choosing the correct calcium assay  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  fluorescent calcium assay kits to help facilitate your research.

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Calcium Assay Kits

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 Calcium assay kits are supplied in a variety of sizes ranging from 2 plates to the largest-scale HTS campaign (>100 plates).

Calcium flux assay data acquired using ION's ratiometric calcium kit. Dose response to ionomycin, compared to FLIPR kit. GPCR assay, muscarinic receptor, showing a dose response to carbachol.
Sample data showing multiplexing with GFP cells using Brilliant Calcium Gold outperforms fluo-4 in a GPCR assay.
GPCR assay, purinergic receptor, kinetic fluorescence profile of calcium mobilization upon stimulation with ATP

Calcium indicators

Want to read more about our diverse collection of fluorescent calcium indicators, click here.

Red calcium indicator, ICR-1, that's ideal for multiplexing, optogenetics, and photopharmacology.
Images of Fluo-4 loaded HeLa cells before and after the addition of ionomycin, a calcium ionophore.
A red-shifted calcium indicator as shown in excitation and emission spectra and representative fluorescence images in a calcium mobilization assay

Additional reagents included in all kits for improving indicator loading and, retention, and reducing extracellular fluorescence

Although the calcium 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.

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.

TRS is a masking solution that is composed of a membrane impermeable fluorescence quencher that reduces extracellular fluorescence. Inclusion of this reagent ensures the fluorescence you are measuring is coming from within your cells. Use of this reagent is not recommended for cell suspensions.

Be Brilliant! Explore our line of Calcium Assays and Indicators

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Image Products Excitation Emission Kd Price Quantity Action
Brilliant calcium assay flex packaging, red background
Brilliant Calcium
High-throughput, no wash calcium (Ca²⁺) assay. An optimal solution for measuring intracellular Ca²⁺ dynamics, including effectors such as GPCRs and protein transporters.
$182.00 – $3,069.00
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Brilliant calcium gold, calcium mobilization assay product packaging
Brilliant Calcium Gold
ION Biosciences Brilliant Calcium Gold Assay is a total assay solution for multi-well plate-based, high-throughput measurements of Ca²⁺ mobilization mediated through a wide-variety of plasma membrane and intracellular calcium channels. The kit includes our Fluo-Gold dye (Ex/Em: 525/550 nm), which is red-shifted for compatibility with GFP and other green fluorescent indicators.
$182.00 – $399.00
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Ratiometric calcium assay kit, using Fura-2 AM, packaging
ION Essentials Ratiometric Calcium
Combines Fura-2 AM with the necessary reagents for conducting no wash, ratiometric calcium flux assays compatible with plate reader and fluorescence microscopy applications. Individual components are provided to give you the flexibility needed to customize your assay.
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$170.00 – $374.00
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