Calcium indicator forms and formats 

We provide our 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.

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 kits are supplied in a variety of sizes ranging from trial packs to the largest-scale HTS (inquire).

(EX 525 nm, EM 550 nm, Kd 400nM) Brilliant Calcium Gold 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 our Fluo-Gold dye, which is red-shifted for compatibility with GFP and other green fluorescent indicators. 10 plates

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.  

(EX 525 nm/Em 550 nm, Kd 400 nM) Fluo-Gold 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.

Dual wavelength calcium 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.

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, we also offer 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 our Ca²⁺-sensitive fluorescent indicators.