Fluo-8® Calcium Reagents and Screen Quest™ Fluo-8 NW Calcium Assay Kits

Introduction

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Calcium acts as a universal second messenger in a variety of cells. The beginning of life, the act of fertilization, is regulated by Ca²⁺. Numerous functions of all types of cells are regulated by Ca²⁺ to a greater or lesser degree. Since the 1920s, scientists have attempted to measure Ca²⁺, but few were successful due to limited availability of Ca²⁺ probes. The first reliable measurements of Ca²⁺ were performed by Ridgway and Ashley by injecting the photoprotein aequorin into the giant muscle fiber of the barnacle. Subsequently, in the 1980s, Tsien and colleagues produced a variety of fluorescent indicators. Among them the fluorescein-based Ca²⁺ reagents (such as Fluo-3 and Fluo-4) have provided trustworthy methods for measuring Ca²⁺. Since the development of these Ca²⁺ probes, investigations of Ca²⁺ -related intracellular phenomena have skyrocketed.

Fluo-8^® Calcium Indicators, the Brightest Calcium Dyes

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Since being introduced, Fluo-3 imaging and its analogs (such as Fluo-4) have revealed the spatial dynamics of many elementary processes in Ca²⁺ signaling. Fluo-3 and Fluo-4 have also been extensively used for flow cytometry and microplate-based (such as FLIPR™) calcium detections. However, the weak signal and harsh dye-loading conditions have limited their applications in some cellular analysis. Our Fluo-8^® serial calcium detection reagents have been developed to address these limitations of Fluo-3 and Fluo-4.

The most important properties of Fluo-3 and Fluo-4 in cellular applications are their absorption spectrum compatible with excitation at 488 nm by argon-ion laser sources, and a very large fluorescence intensity increase in response to Ca²⁺ binding. These two valuable properties have been retained intact with our Fluo-8^® Ca²⁺ detection reagents. The absorption and emission peaks of Fluo-8^® reagents are 490 nm and 514 nm, respectively. They can be well excited with an argon ion laser at 488 nm, and their emitted fluorescence (at wavelength 514 nm) increases with increasing Ca²⁺ . Fluo-8^® is determined to undergo a > 200-fold increase in fluorescence upon binding to Ca²⁺ . Because the range of increase in Ca2+ in many cells after stimulation is generally 5- to10-fold, Fluo-8^® is an excellent probe to use with high sensitivity in this region. The Kd of Fluo-8^® is estimated to be 389 nM (22°C, pH 7.0–7.5), but this value may be significantly influenced by pH, viscosity, and binding proteins in vivo conditions.

Besides their convenient 488 nm excitation wavelength and large fluorescence enhancement by calcium, Fluo-8^® is much brighter in cells than Fluo-3 and Fluo-4 as shown in Figure 1. In addition, Fluo-8^® is much more readily loaded into live cells than Fluo-3 and Flu-4, both of which require 37°C for optimal cell loading. Fluo-8^® reagents have a less temperature-dependent cell loading property, giving similar results either at room temperature or 37°C. This characteristic makes Fluo-8^® more robust for HTS applications.

Compared to Fluo-3 and Fluo-4, our Fluo-8^® calcium detection reagents have the following advantages:

• Convenient Wavelengths: Maximum excitation @ ~490 nm; maximum emission @ ~514 nm.
• Enhanced Intensity: 2 times brighter than Fluo-4 AM; 4 times brighter than Fluo-3 AM.
• Faster Loading: dye loading at room temperature (rather than 37ºC that is required for Fluo-4 AM).
• Versatile Ca²⁺ -Binding K_d as shown in Table 1.
• Versatile Packing Sizes to Meet Your Special Needs: 1 mg; 10x50 µg; 20x50 µg; HTS packages.

Use of Fluo-8^® AM Esters

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1. Load Cells with Fluo-8^® AM Esters: AM esters are the non-polar esters that readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. AM esters are widely used for loading a variety of polar fluorescent probes into live cell non-invasively. However, cautions must be excised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted just before use in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions may be stored desiccated at –20°C and protected from light. Under these conditions, AM esters should be stable for several months.
   Following is our recommended protocol for loading Fluo-8^® AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.
   1. Prepare a 2 to 5 mM stock solution of Fluo-8^® AM esters in high-quality, anhydrous DMSO.
   2. On the day of the experiment, either dissolve Fluo-8^® in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a working solution of 1 to 10 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.02% Pluronic^® F-127. For most of cell lines, Fluo-8^® reagents with a concentration ranging from 4-5 uM are recommended. The exact concentration of the indicator required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.
      Note: The nonionic detergent Pluronic^® F-127 is sometimes used to increase the aqueous solubility of Fluo-8® AM esters. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
   3. If your cells containing the organic anion-transports, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators.
      Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest.
   4. Add equal volume of the dye working solution (from Step b or c) into your cell plate.
   5. Incubate the dye-loading plate at a cell incubator or room temperature for 20 minutes to one hour.
      Note: Decreasing the loading temperature might reduce the compartmentalization of the indictor.
   6. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 2.5 mM probenecid, if applicable) to remove excess probes.
   7. Run the experiments at Ex/Em = 490/525 nm
2. Measure Intracellular Calcium Responses: see figure 1.

Use of Screen Quest™ Fluo-8 NW Calcium Assay Kits for HTS Applications

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GPCR activation can be detected by direct measurement of the receptor mediated cAMP accumulation, or changes in intracellular Ca²⁺ concentration. GPCR targets that couple via Gq produce an increase in intracellular Ca²⁺ that can be measured using a combination of Fluo-8® reagents and a fluorescence microplate reader. The fluorescence imaging plate readers (such as, FLIPR™, FDSS or BMG NovoStar™) have a cooled CCD camera imaging system which collects the signal from each well of a microplate (both 96 and 384-well) simultaneously. These plate readers can read at sub-second intervals, which enables the kinetics of the response to be captured, and has an integrated pipettor that may be programmed for successive liquid additions. Besides their robust applications for GPCR targets, our Screen Quest™ Fluo-8 Calcium Assay Kits can be also used for characterizing calcium ion channels and screening calcium ion channel-targeted compounds.

Compared to other commercial calcium assay kits that either based on Fluo-3 or Fluo-4, our Screen Quest™ Calcium Assay Kits have the following advantages for HTS applications:

• Broad Applications: work with both GPCR and calcium channel targets.
• Convenient Spectral Wavelengths: maximum excitation @ ~490 nm; maximum emission @ ~514 nm.
• Flexible Dye Loading: dye loading at room temperature (rather than 37ºC required for Fluo-4 AM).
• No Wash Required and No Quencher Interference with Your Targets.
• Robust Performance: enable calcium assays that are impossible with Fluo-4 AM or Fluo-3 AM.
• Strongest Signal Intensity: 2 times brighter than that of Fluo-4 AM; 4 times brighter than that of Fluo-3 AM.

 

Use of Fluo-8^® Salts

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Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in fluorescence microplate readers) of the indicators in solutions with precisely known free Ca²⁺ concentrations. Calibration solutions can be used based on 30 mM MOPS EGTA Ca²⁺ buffer. In general, water contains trace amount of calcium ion. It is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca²⁺ concentrations

1. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA
2. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl₂

To determine either the free calcium concentration of a solution or the K_d of a single-wavelength calcium indicator, the following equation is used:

[Ca]_free = K_d[F ─ F_min]/F_max ─ F]

Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, F_min is the fluorescence intensity in the absence of calcium and F_max is the fluorescence intensity of the calciumsaturated probe.

The dissociation constant (K_d) is a measure of the affinity of the probe for calcium. The calcium-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ response calibrations of intracellular indicators typically yield K_d values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca²⁺ buffers in the presence of ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin or Triton^® X-100 can be used to expose the indicator to the controlled Ca²⁺ levels of the extracellular medium. The Kd values of Fluo-8^® reagents are listed in Table 1 for your reference.

Conclusions

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Because of the importance of Ca²⁺ in biology, numerous techniques/methods for analyzing the mechanisms of cellular and/or subcellular Ca²⁺ activity have been established. Unfortunately, however, there is no one best technique/method with which one can measure Ca²⁺. Although each method for analyzing Ca²⁺activity has certain advantages over the others, each also suffers drawbacks. With the outstanding properties described above, we believe that Fluo-8^® calcium detection reagents and Screen Quest™ Fluo-8NW Calcium Assay Kits provide new powerful tools for intracellular calcium analysis and monitoring in a variety of biological systems in coupling with the rapid advance in fluorescence instrumentation.

As might have been predicted, the interests of many researchers shifted from Ca²⁺ analysis at the cellular level to the subcellular level. It has been found that Ca2+ is not even distributed throughout the whole cell and that intracellular heterogeneity of Ca²⁺ (such as Ca²⁺ waves and Ca²⁺ sparks) is observed in a variety of cells (e.g., oocyte, heart muscle cell, hepatocyte, and exocrine cell). With the advent of the confocal laser scanning microscope (CLSM) in the 1980s and advanced microplate readers in 2000s (such as FLIPR, FDSS and NOVOStar dedicated for intracellular Ca²⁺ detections), measurement of intracellular Ca²⁺ has accelerated significantly. Confocal laser scanning microscopy, and more recently multiphoton microscopy, allows the precise spatial and temporal analysis of intracellular Ca²⁺ activity at the subcellular level in addition to the measurement of its concentration.

Ordering Information

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