ION Biosciences ‒ Fluorescent Ion Indicators & Assay Kits

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

Fluorescent Calcium Indicators

Extensive research, including seminal contributions by ION’s principal scientists, 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, 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 Calcium 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.

Calcium Indicator Forms and Formats

ION Biosciences provides their calcium dyes in a number of formats to best suit the intended application including membrane permeable forms, membrane impermeable forms and indicators packaged with other key reagents into calcium assay kits that are optimized and validated to ensure excellent performance.

Single Wavelength Calcium 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.

• Fluo-4 (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.
• Fluo-3 (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.
• Fluo-2 (EX 490 nm/Em 515 nm, Kd 290 nM), also known as Fluo-2 HA, 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.
• Fluo-Gold (EX 525 nm/Em 550 nm, Kd 400 nM) is a gold fluorescent, intracellular calcium indicator; designed to measure intracellular calcium flux in high throughput screening and fluorescence microscopy applications. Fluo-Gold’s red-shifted excitation and emission enable multiplexing with GFP-expressing cells or other green fluorescent indicators and minimizes interference from green-fluorescent compounds.
• ICR-I (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.

Ratiometric Calcium Indicators

These indicators are particularly useful when more accurate quantification 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 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.

• Fura-2: (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.
• Fura-2 LR: (Ex 340/380 nm/505 nm, Kd 145 nM) with spectral properties identical to Fura-2. ION Biosciences also offers Fura-2 LR, a leakage-resistant variant. Fura-2 LR is better retained in some cell lines and may provide superior results in cases where intracellular dye retention is needed.

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 their Ca²⁺-sensitive fluorescent indicators.

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 their 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.

• Brilliant Calcium Flex provides all necessary reagents in a format to maximize flexibility for your assay design and customization challenges. Brilliant Calcium Flex (EX 490 nm, EM 515 nm) is a high-throughput, no wash calcium mobilization assay. The most flexible solution for measuring the activity of GPCRs coupled to intracellular Ca²⁺ release. 10 plates.
• Brilliant Calcium Gold (EX 525 nm, EM 550 nm, Kd 400nM) is a calcium flux assay for multi-well plate-based, high throughput measurements of calcium mobilization mediated through a wide variety of plasma membrane and intracellular calcium channels. The kit includes Ion’s Fluo-Gold dye, which is red-shifted for compatibility with GFP and other green fluorescent indicators. 10 plates.
• Ratiometric Calcium Essentials kit provides 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 the user the flexibility needed to customize their assay.
  • EX 340/380 nm, EM 505 nm, Kd 145nM)
  • High-throughput, no wash ratiometric calcium (Ca²⁺) assay.
  • No wash, compatible with plate reader and fluorescence microscopy applications.

Potassium indicators are molecules that exhibit an increase in fluorescence upon binding K⁺.

Potassium (K⁺) in Physiology

Potassium, along with sodium (Na⁺), is one of nature’s most important monovalent metal cations. Ion channels and transporters for K⁺ play a diverse range of functions from modulating neuronal activity to regulating solute balance. Potassium channels are by far the largest and most structurally diverse class of ion channels and much remains unknown about their functions in normal physiology and disease. An increasingly long list of K⁺ channels and K⁺ transporters are being associated with a wide variety of disorders.

Potassium channel and K⁺ transporter-targeted drugs already provide effective treatments for a wide breadth of indications including epilepsy, autism, type-2 diabetes, and diuresis. Unfortunately, K⁺ channels, particularly the Kv11.1 channel (a.k.a. hERG), may be the unintended target of other therapeutics resulting in sometimes deadly side-effects. As a result, pre-clinical testing of drug candidates to determine their effects on hERG channel activity is required.

Because expanding knowledge of K⁺ channels and K⁺ transporters is of such intense interest to both basic and drug discovery researchers, a facile means to measure their activity is critical. Unfortunately, measuring intracellular and extracellular K⁺ levels and dynamics using fluorescent indicators is challenging. In most cells at rest, cytoplasmic K⁺ concentrations are above 100 mM while Na⁺ concentrations are in the 3 – 5 mM range. Conversely, extracellular K⁺ concentrations are typically in the 3-5 mM range while Na⁺ concentrations are above 100 mM. Therefore, an effective K⁺sensor needs to be able to discriminate between K⁺, Na⁺ and other common physiological cations while possessing a K⁺ affinity appropriate to detect physiologically relevant changes in K⁺ concentrations.

Fluorescent Potassium Indicators

Extensive research has afforded the ION Potassium Green (IPG) family of excellent K⁺ sensors, formerly known as Asante Potassium Green (APG). These molecules are small, synthetic fluorochromes which incorporate a K⁺-binding moiety. Under conditions where K⁺ is not bound, the fluorescence of the sensor is significantly quenched. When K⁺ is bound, the quenching is relieved, and the fluorescence of the sensor dramatically increases.

The IPG family of indicators are the best-in-class replacements for the legacy K⁺ indicator, PBFI. Unlike PBFI, the spectral properties of the IPG family of K⁺ indicators make them convenient to use with common filter sets (e.g. YFP and FITC) as well as multiphoton approaches, and the range of K⁺ affinities make them useful for applications as diverse as extracellular K⁺ sensing to monitoring intracellular K⁺ dynamics. The IPG family of K⁺ indicators is compatible with a wide variety of detectors including fluorescent microscopes, plate readers, flow cytometers, and fluorescent indicator-doped solid-state sensors.

• IPG-1 (EX 525 nm, EM 545 nm, Kd 50 mM) has the lowest affinity of the ION Potassium Green indicators. It is the indicator that is best positioned to respond to modest changes in intracellular K⁺ concentrations resulting from activation of plasma membrane K⁺ channels.
• IPG-4 (EX 525 nm, EM 545 nm, Kd 7 mM) has the highest K⁺ affinity of Ion’s single-wavelength indicators making it appropriate for conditions where there may be large changes in intracellular K⁺ (e.g. highly active synapses) or as an extracellular K⁺ sensor.
• IPG-2 (EX 525 nm, EM 545 nm, Kd 18 mM) is the ION Potassium Green indicator with intermediate affinity. Currently, IPG-2 is ION’s most popular intracellular K⁺ indicator.
• PBFI (EX 340/380 nm, EM 505 nm, Kd 4 mM); PBFI, AM is a potassium indicator that is ratiometric and UV light-excitable. This acetoxymethyl (AM) ester form is useful for noninvasive measurements of intracellular potassium levels.

Thallium (Tl⁺)-Sensitive Fluorescent Indicators

While the IPG family of dyes are excellent fluorescent K⁺ indicators, the challenges of discriminating between K⁺ and Na⁺ are not trivial, and together with the often modest changes in intracellular or extracellular K⁺ results in relatively small signals compared to those observed with the Ca²⁺-sensitive fluorescent indicators, such as Fluo-4. In many instances, the Tl+ flux assay offers an outstanding alternative to K⁺-sensitive fluorescent indicators. The Tl⁺ flux assay takes advantage of a Tl⁺-selective fluorescent indicator, Thallos, and the fact that K⁺ channels and K⁺ transporters readily accept Tl⁺ as a K⁺ surrogate. When Tl⁺ is added to the outside of cells loaded with Thallos, Tl⁺ entering cells through K⁺ channels and K⁺ transporters results a dramatic increase in fluorescence. Today, the Tl⁺ assay is the most commonly used approach for large-scale high-throughput screening (HTS) of K⁺ channels and K⁺ transporters.

• Thallos (EX 490 nm, EM 515 nm) is the best-in-class Tl⁺-sensitive fluorescent indicator. It offers outstanding cell loading and excellent compatibility with commonly available FITC filters.
• Thallos Gold (EX 530 nm, EM 550 nm) is a gold fluorescent intracellular thallium (Tl+) indicator. Red-shifted excitation and emission enable multiplexing with GFP-expressing cells or other green fluorescent indicators.

Potassium Channel Assays Using Thallium Flux

Indicator Forms and Formats

ION Biosciences provides their IPG family of K⁺-sensitive fluorescent indicators in membrane permeable and membrane impermeable forms. We offer Thallos as part of Ion’s convenient Brilliant Thallium Assay kits and, unlike other vendors, as a stand-alone product for investigators who prefer to use their own solutions and reagents.

Cell Permeable Acetoxymethyl Group (AM) Esters

The native forms of K⁺-sensitive and Tl⁺-sensitive fluorescent indicators are negatively charged and membrane impermeable. However, masking the negative charge using non-polar, ester-linked moieties (AM esters) allow the molecules to enter cells through passive diffusion. 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 and Retention

Although the AM forms of ION’s fluorescent K⁺ and Tl⁺ 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 K⁺ and Tl⁺ 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 K⁺-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 their K⁺-sensitive fluorescent indicators.

Sodium and thallium indicators, which exhibit an increase in fluorescence upon binding Na⁺ or Tl⁺, can be used to measure the activity of sodium channels and transporters.

Sodium (Na⁺) in Physiology

Sodium, along with potassium (K⁺), is one of the most important monovalent metal cations in living organisms. Sodium channels, Na⁺-permeable non-selective monovalent cation channels, and Na⁺ transporters play critical roles including modulating neuronal activity, powering transport of nutrients and signaling molecules, and regulating solute balance. Na⁺-permeable channel and Na⁺ transporter-targeted drugs provide effective treatments for a diversity of indications: epilepsy, pain, bipolar disorder, depression, diuresis, and many others. An increasingly long list of Na⁺-permeable channels, and Na⁺ transporters are being associated with a wide variety of disorders. As a result, interest in Na⁺-permeable channels and Na⁺ transporters as drug targets remains high.

Because expanding the understanding of Na⁺ permeable channels and Na⁺ transporters is of such intense interest to both basic and drug discovery researchers, a facile means to measure their activity is required. Unfortunately, measuring intracellular and extracellular Na⁺ levels and dynamics using fluorescent indicators is challenging. In most cells at rest, cytoplasmic Na⁺ concentrations are ~5 mM while K⁺ concentrations are above 100 mM. Conversely, extracellular Na⁺ concentrations are typically >100 mM while K⁺ concentrations are in the 3-5 mM range. Therefore, an effective Na⁺ sensor needs to be able to discriminate between K⁺, Na⁺ and other common physiological cations while possessing an appropriate Na⁺ affinity to detect physiologically relevant changes in Na⁺ concentrations.

Sodium Channel Assays and Sodium Indicators

• ION Natrium Green-2 (ING-2), formerly known as Asante Natrium Green (ANG-2), is a small, synthetic fluorochrome fused with a Na⁺-binding moiety. Under conditions where Na⁺ is not bound, the fluorescence of the sensor is significantly quenched. When Na⁺ is bound, the quenching is relieved, and the fluorescence of the sensor dramatically increases. ING-2 (EX 525 nm, EM 545 nm, Kd 20 mM) is the best-in-class fluorescent Na⁺ sensor replacing previous Na⁺ indicators such as SBFI and CoroNa Green. Its affinity for Na⁺ is well suited to respond to changes in intracellular Na⁺ concentrations resulting from activation of plasma membrane Na⁺ permeable channels and Na⁺ transporters. It is compatible with a wide variety of detectors including fluorescent microscopes, plate readers, and flow cytometers using common filter sets like sets (e.g. YFP and FITC) as well as multiphoton approaches. ING-2 has also proven useful for high-throughput screening (HTS).
• ING-1 (EX 525 nm, EM 545 nm, Kd 92 mM) is a yellow-green fluorescent, sodium indicator with a lower affinity for sodium than ING-2.
• SBFI is a UV-excitable, ratiometric green indicator for intracellular sodium (Na⁺) measurements. Ex/Em: 340/505 nm can be used to measure Na⁺-bound SBFI, and Ex/Em: 380/505 nm can be used to detect Na⁺-free SBFI. It is ~18X more selective for Na⁺ over K⁺. Ratiometry is optimal for imaging applications where quantification of intracellular Na⁺ concentrations is desired, and reduces effects of photobleaching, heterogenous dye loading, and variable cell morphology.
• Brilliant Sodium is the first kit designed to measure intracellular sodium in an HTS-compatible format, so you can discover and characterize the effects of many tens of thousands of compounds and environmental factors on effectors of sodium channels and transporters.

Thallium Flux Assays and Thallium Indicators

While ING-2 is an excellent Na⁺-sensitive, fluorescent indicator, the challenges of discriminating between K⁺ and Na⁺ are not trivial, and together with the often modest changes in intracellular Na⁺ results in relatively small signals compared to those observed with the Ca2⁺-sensitive fluorescent indicators, such as Fluo-4.

In many instances, the thallium flux assay offers an outstanding alternative to ING-2 and other Na⁺-sensitive fluorescent indicators. The Tl⁺ flux assay takes advantage of a Tl⁺-selective fluorescent indicator, Thallos, and the fact that sodium channels and transporters readily accept Tl⁺ as a Na⁺ surrogate. When Tl⁺ is added to the outside of cells loaded with Thallos, Tl⁺ entering cells through Na⁺ permeable channels and Na+ transporters results in a dramatic increase in fluorescence. Although the Tl⁺ assay is most commonly associated with high-throughput screening (HTS) of K⁺ channels, it is equally suitable for HTS of sodium channels and transporters.

Sodium Channel Assays Using Thallium Flux

• Thallos, (EX 490 nm, EM 515 nm) is the best-in-class Tl⁺-sensitive fluorescent indicator. It boasts outstanding cell loading and excellent compatibility with a wide variety of detectors including fluorescent microscopes, plate readers, and flow cytometers using commonly available FITC filters.
• Brilliant Thallium Flex (EX 490 nm, EM 515 nm) is a high-throughput, no wash thallium flux assay. An optimal solution for measuring the activity of potassium channels and transporters, as well as sodium channels, non-selective cation channels, and GPCRs. 10 plates.
• Brilliant Thallium Gold (EX 530 nm, EM 550 nm) includes Thallos Gold – ION’s gold fluorescent thallium (Tl+) indicator. Red-shifted excitation and emission enable multiplexing with GFP-expressing cells or other green fluorescent indicators. Useful for fluorescence-based potassium channel assays and for studying a wide variety of monovalent cation channels (sodium channels), transporters, and GPCRs.
• Brilliant Thallium Snapshot (EX 490 nm, EM 515 nm) is a thallium flux assay adaptation designed to work with standard plate readers, microscopes, flow cytometers, and more. For measuring flux through potassium (K⁺), sodium (Na⁺), and non-selective cation channels and their effectors including transporters and GCPRs. 10 plates.

Indicator Forms and Formats

We provide ING-2 in membrane permeable and membrane impermeable forms, as well as in Brilliant Sodium Assays. We offer Thallos as part of the convenient Brilliant Thallium Assay kits and, unlike other vendors, as a stand-alone product for investigators who prefer to use their own solutions and reagents.

Cell Permeable, Acetoxymethyl Group (AM) Esters

The native forms of the Na⁺-sensitive and Tl⁺-sensitive fluorescent indicators are negatively charged and membrane impermeable. However, masking the negative charge using non-polar, ester-linked moieties (AM esters) allow the molecules to enter cells through passive diffusion. 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, Na⁺ and Tl⁺ 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 Na⁺ and Tl⁺ indicators into cells, it is highly recommended.

Another factor that can affect cell loading and intracellular localization is the presence of anion transport proteins in both plasma and intracellular membranes. 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 Na⁺ and Tl⁺ sensitive fluorescent indicators nearly impossible. Fortunately, probenecid, an organic anion transport inhibitor can be an effective means of inhibiting the transporters, improving cytoplasmic localization and indicator retention. ION offers a convenient, 100X probenecid solution that may be used in conjunction with Na⁺ and Tl⁺-sensitive fluorescent indicators. While probenecid is generally well-tolerated over the time course of typical experiments using Na⁺ and Tl⁺-sensitive fluorescent indicators, effects on the activity of TRP channels has been reported.

Thallium can be used as a surrogate ion to measure the activity of potassium and sodium channels and transporters.

Thallium Assays and Indicators

Sodium (Na⁺) and potassium (K⁺) are the two most physiologically important monovalent metal cations. Transporters and ion channels for Na⁺ and K⁺ are found in every cell of every known organism and are critical for life. While drugs targeting these channels and transporters are used to treat indications ranging from type II diabetes, to epilepsies, to blood pressure, the therapeutic potential of this incredibly diverse set of proteins remains largely unknown. Therefore, ion channel assays are essential to promote the rapid discovery and evaluation of monovalent cation channels and transporters as potential therapeutic targets.

While ION’s class-leading sodium and potassium-sensitive fluorescent indicators are excellent tools for studying cellular Na⁺ and K⁺ dynamics, the relatively small changes in the intracellular concentrations of Na⁺ and K⁺ under normal physiological conditions can make Na⁺ and K⁺ indicators challenging to use, particularly for high-throughput screening (HTS) applications. In many instances, the thallium flux assay offers an outstanding alternative to fluorescent potassium and sodium indicators.

The thallium flux assay takes advantage of a Tl⁺-selective fluorescent indicator, Thallos, and the fact that potassium channels and transporters, as well as sodium channels and transporters, readily accept Tl⁺ as surrogate cation. When Tl⁺ is added to the outside of cells loaded with Thallos, Tl⁺ entering cells through ion channels and transporters permeable to Na⁺ or K⁺ results in a dramatic increase in fluorescence. Although the Tl+ flux assay is the most commonly associated with HTS of potassium channels, it is equally suitable for HTS of sodium channels and sodium transporters, as well as non-selective, monovalent cation channels.

Thallium Indicator Forms and Formats

Thallos is a small, synthetic fluorochrome which incorporates a Tl⁺-binding moiety with excellent selectivity for Tl⁺ over most other physiologically relevant metal cations (e.g. Ca²⁺). Under conditions where Tl⁺ is not bound, the fluorescence of the sensor is significantly quenched. When Tl⁺ is bound, the quenching is relieved, and the fluorescence of the sensor dramatically increases.

Thallos is compatible with a wide variety of detectors including fluorescent microscopes, plate readers, flow cytometers. Over the last 20 years, since Dr. David Weaver invented the Tl⁺ flux assay, Tl⁺-sensitive indicators have found numerous uses including high-throughput screening (HTS) of potassium channel, sodium channel, other monovalent cation channels, some monovalent cation transporters, and the Gi/o class of G protein-coupled receptors (GPCR).

In addition to dye, ION Biosciences also offer a selection of Brilliant Thallium Assay kits with individually packaged reagents to provide you with the ultimate flexibility to optimize your ion channel assays. The Brilliant Thallium Snapshot assay is designed to make thallium flux assays more accessible, enabling you to run functional ion channel screens without automation and on readily available, fluorescence-capable instruments.

• Thallos (EX 490 nm, EM 515 nm) is the best-in-class Tl⁺-sensitive fluorescent indicator. It boasts outstanding cell loading and excellent compatibility with a wide variety of detectors including fluorescent microscopes, plate readers, and flow cytometers using commonly available FITC filters.
• Brilliant Thallium Gold (EX 530 nm, EM 550 nm) includes Thallos Gold – ION’s gold fluorescent thallium (Tl⁺) indicator. Red-shifted excitation and emission enable multiplexing with GFP-expressing cells or other green fluorescent indicators. Useful for fluorescence-based potassium channel assays and for studying a wide variety of monovalent cation channels (sodium channels), transporters, and GPCRs. 10 plates.
• Brilliant Thallium Gold Snapshot is a multi-instrument compatible assay solution for well plate-based, high-throughput measurements of thallium (Tl⁺) flux through potassium (K⁺), sodium (Na⁺), and non-selective cation channels, as well as some Na⁺ or K⁺ transporters. Ion’s patent-pending Snapshot technology generates a long-lasting signal so you can easily detect and quantify changes in ion channel or transporter activity on most fluorescence-capable instruments. The inclusion of Thallos Gold AM, a red-shifted thallium indicator, makes this kit the ideal option when using GFP-expressing cells to run potassium channel assays.
• Brilliant Thallium Flex (EX 490 nm, EM 515 nm) is a high-throughput, no wash thallium (Tl⁺) assay. The most flexible solution for measuring the activity of potassium (K⁺), sodium (Na⁺), and non-selective cation channels and their effectors including transporters and GPCRs. 10 plates.
• Brilliant Thallium Snapshot is a high-throughput, no wash thallium (Tl⁺) Snapshot assay. A thallium flux assay adaptation designed to work with standard plate readers, microscopes, flow cytometers, and more, for measuring flux through potassium (K⁺), sodium (Na⁺), and non-selective cation channels and their effectors including transporters and GPCRs. 10 plates.
• Flow Cytometric Potassium Channel Assay is the first assay solution for single-cell, high-throughput measurements of potassium (K⁺) channel activity. Powered by Ion’s patent-pending Snapshot assay technology, ion channel modulation generates a long-lasting signal so you can easily detect and quantify changes in ion channel or transporter activity using Flow Cytometry. This kit may also work for sodium (Na⁺) channels, non-selective cation channels, and some Na⁺ or K⁺ transporters. 200 assays.

Ion’s best assays for measuring cell viability
and mitochondrial function.

Cell Health

An essential step in early stage, translational research is measuring cytotoxicity. ION’s product portfolio is designed to help you easily capture important parameters of cell health, whether your preferred instrument is a plate reader, fluorescence microscope, or flow cytometer, using a collection of criteria that are indicative metrics of cell viability or death. These criteria include intracellular esterase activity as a marker of cell viability, plasma membrane integrity as a marker of cell death, and mitochondrial membrane potential as an early-stage marker of apoptosis. When used in combination, these assays can provide an informative snapshot of cell health.

Intracellular Esterase Activity

Intracellular enzyme activity and dye retention is a hallmark of viable cells. During cell death, increased plasma membrane permeability causes enzyme leakage and a reduction in activity. Dyes that measure esterase activity are modified with ester-moieties, which help them passively diffuse through cell membranes and render the dyes non-fluorescent. Once inside the cell, ubiquitous esterases present in the cytosol cleave ester-moieties, activating dye fluorescence and trapping the dye within the cell. ION sells Calcein AM in multiple formats (dry or in a prepared solution) and in combination with Ethidium Homodimer I as part of ION Vital – Viability so you can simultaneously detect dead cells.

• Calcein AM (Ex/Em 495 nm/515 nm) is a membrane-permeant, non-fluorescent form of calcein that is converted to green fluorescent calcein in viable cells, resulting in uniform cytosolic fluorescence. Calcein is well retained within the cytosol of most healthy cells with intact cell membranes.
• ION Vital ‒ Viability combines Ethidium Homodimer I and Calcein AM to provide a two-color, fluorescence-based assay to discriminate between live and dead cells.
• BCECF AM (Ex/Em 490 nm/535 nm) is the most popular green fluorescent, intracellular pH indicator, however it can also be used as a marker of intracellular esterase activity. For this application, BCECF should be used in non-ratiometric mode using standard FITC excitation and emission settings.

Plasma Membrane Integrity

A loss of plasma membrane integrity is an indicator of cell death. Nucleic acid-binding, cell impermeant dyes, like Ethidium Homodimer I (EthD-I), can be used to label the nuclei of cells with compromised plasma membranes. These dyes are excluded from intact, healthy cells and exhibit minimal fluorescence in the absence of nucleic acids. Once the dye crosses the cell membrane and binds to nucleic acids (DNA) inside the cell, it displays a significant increase in fluorescence. ION sells EthD-I in multiple formats (dry or in a prepared solution) and in combination with Calcein AM as part of ION Vital – Viability so you can simultaneously detect viable cells.

• Ethidium Homodimer I (Ex/Em: 528 nm/617 nm) is a membrane-impermeable, high-affinity, nucleic acid stain that selectively labels dead cells with compromised plasma membranes.
• ION Vital – Viability combines Ethidium Homodimer I and Calcein AM to provide a two-color, fluorescence-based assay to discriminate between live and dead cells.

Mitochondrial Membrane Potential

Mitochondrial membrane potential is one of the driving forces behind mitochondrial ATP synthesis, and is necessary for maintaining cell homeostasis. During early stages of apoptosis, mitochondrial function and membrane integrity are disrupted by the activation of apoptotic proteins and subsequent release of cytochrome C, mitochondrial Ca2+ stores, and other components. Membrane potential sensitive dyes, such as JC-1 and JC-10, can be used to monitor the loss of mitochondrial membrane polarization observed during apoptosis. These dyes compartmentalize in healthy mitochondria, leading to the formation of aggregates that exhibit orange fluorescence. When mitochondrial membrane potential is lost during apoptosis, these dyes no longer accumulate and return to green fluorescent monomers. ION sells JC-10 individually and as part of their optimized ION Vital Assay line of products.

• ION Vital – MitoVolt is a mitochondrial membrane potential assay kit that uses JC-10, a potentiometric dye that is more soluble than JC-1. It is an ideal solution for detecting changes in mitochondrial membrane potential due to cell apoptosis or other stress-inducing phenomena, and is compatible with fluorescence microscopy, flow cytometry, and plate reader applications.
• JC-10 (Ex/Em: 490,540 nm/525, 590nm) is a mitochondrial membrane potential probe. It possesses superior aqueous solubility compared to its better-known analogue, JC-1. At low concentrations, JC-10 is monomeric and emits a green fluorescence. JC-10 accumulates in healthy mitochondria, forming J-aggregates that exhibit an orange fluorescence. Its potentiometric behavior allows for ratiometric analysis of mitochondrial membrane potential, where a shift from orange to green fluorescence is indicative of compromised mitochondria.