| Overview |
 Printer Friendly Version
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| Ex/Em (nm) | 494/517 |
| MW | 1046.93 |
| CAS # | 1345980-40-6 |
| Solvent | DMSO |
| Storage | F/D/L |
| Category |
GPCR Calcium GPCR Assays |
| Related |
Calcium Channels pH and Ion Indicators |
- Enhanced Intensity → 2x brighter than Fluo-4 AM
- Better & Faster Loading → Room temperature instead of 37°C
- More Robust Results → Probe effective in a wide range of temperatures
- Variety of Kd → Many options for calcium binding dissociation constant
The History
With hundreds of thousands of peer-reviewed papers published annually, it is clear that there is both intense and widespread interest in calcium and its function in biological systems. This is because calcium ions (Ca2+) play a crucial role in a number of biological processes, ranging from signal transduction to muscle contraction to neurotransmitter regulation. This ubiquity of calcium, and the subsequent need to study it, has prompted extensive effort into the development of stable, sensitive and convenient calcium indicators, as researchers looked for tools to help quantitate and visualize intracellular calcium.
The first big breakthrough came in the 1980s, when a group of researchers at UC Berkeley developed a calcium indicator based on fluorescein. Their research laid the groundwork for now well-established calcium probes such as Fluo-3 AM and Fluo-4 AM. These fluorescein-based AM esters readily pass through the membranes of living cells, where once inside, they are rapidly hydrolyzed by esterases and set to detect calcium. Upon successful binding with calcium, Fluo-3 or Fluo-4 will become fluorescent, emitting a signal at ~520 nm when excited by a ~490 nm light source.
While a significant achievement, the first generation of fluorescein-based dyes suffered from several major problems, specifically with loading conditions and brightness. Cells had to be treated with the fluorescein-based AM ester under harsh conditions that could damage the integrity of the sample. And if loading was not conducted under just perfect conditions, signal quality and intensity would be dramatically diminished. Such prominent deficiencies should not exist in a fluorescent probe, particularly one investigating a substance as vital to biology as calcium. It is with this thought in mind that AAT Bioquest set out to develop a new fluorescein-based calcium indicator. And after much research, development and optimization, we are pleased to announce the next generation of fluorescein-based dyes, headed by our flagship indicator, Fluo-8® AM.
Fluo-8® AM Features
Fluo-8® AM is a novel fluorescent green calcium indicator that builds upon the fluorescein core while rectifying the issues that plagued earlier indicators like Fluo-3 AM and Fluo-4 AM. In particular, it has several key advantages:
- Enhanced Intensity - Tests have shown Fluo-8® AM is twice as bright as Fluo-4 AM and up to four times brighter than Fluo-3 AM
- Better Loading - Fluo-8® AM can be loaded at room temperature, unlike the 37°C incubation required by Fluo-4 AM
- Faster Loading - Room temperature loading means no need to warm up and cool down cells; finish loading in as little as 20 minutes
- Robust - Fluo-8® is less temperature dependent than other probes, leading to more consistent results
- Versatile Kd - Fluo-8® AM is available with a variety of Ca2+ binding dissociation constants, see Table 1 for details
As with its predecessors, Fluo-8® AM is completely compatible with use in flow cytometry, fluorescence microscopy, fluorescence spectroscopy and the fluorescence microplate platform. To read more about how Fluo-8® AM has revolutionized calcium imaging, or to see additional Fluo-8® product applications, please click here.
Table 1. Ca2+ Binding Dissociation Constants
| Product | Excitation | Emission | Ca2+ Kd | Cat No. | Pricing |
| Fluo-8® AM | 490 nm | 514 nm | 389 nM | 21080 | $245 for 1 mg |
| Fluo-8H™ AM | 490 nm | 514 nm | 232 nM | 21090 | $295 for 1 mg |
| Fluo-8L™ AM | 490 nm | 514 nm | 1.86 µM | 21096 | $295 for 1 mg |
| Fluo-FF™ AM | 494 nm | 514 nm | 10 µM | 21105 | $295 for 1 mg |
| Spectrum | Advanced Spectrum Viewer |
Use of Fluo-8® AM Esters
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.
a) Prepare a 2 to 5 mM stock solution of Fluo-8® AM esters in high-quality, anhydrous DMSO.
b) 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.
c) 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.
d) Add equal volume of the dye working solution (from Step b or c) into your cell plate.
e) 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.
f) 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.
g) Run the experiments at Ex/Em = 490/525 nm
Use of Screen Quest™ Fluo-8 NW Calcium Assay Kits for HTS Applications
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.
Figure 2. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of the Screen Quest™ Fluo 8-NW calcium assay kit and Fluo-4 NW kit (according to the manufacturer’s instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG LabTech) to achieve the final indicated concentrations. The EC50 of Fluo-8 NW is about 1.2 uM.
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 37 ºC 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
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
A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA
B. 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 Kd of a single-wavelength calcium indicator, the following equation is used:
[Ca]free = Kd[F ─ Fmin]/Fmax ─ F]
Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, Fmin is the fluorescence intensity in the absence of calcium and Fmax is the fluorescence intensity of the calcium-saturated probe.
The dissociation constant (Kd) 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 Kd 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.
| References & Citations |
 Citation Explorer
|
Below, you may find a small sampling of specific Fluo-8® AM applications sorted by field of study. To inquire about a potential application of Fluo-8® AM, or to consult with our fluorescent dye specialists, please contact us at support@aatbio.com or 1-800-990-8053.
In Oncology, Fluo-8® AM has been used to study:
» Breast cancer cells by monitoring intracellular Ca2+ flux associated with apoptosis and inhibition by 2-aminoethoxydiphenyl borate[1]
» Antitumor activity by way of thioredoxin-binding protein 2 and its dependence on intracellular calcium concentration[2]
» Bcl-1 and Bcl-2 regulation through characterization of cytosolic transport as quantified by calcium flux[3]
» Ca2+ influx and Ca2+ channel activity in NCI-H460 cells as a parameter for monitoring progression of non-small cell lung cancer[4]
» Ca2+ release by HN4 cells and CLIC4 upregulation of apoptosis through mitochondrial and endoplasmic reticulum pathways[5]
In Cardiology, Fluo-8® AM has been used to study:
» Low-energy far-field stimulation as a therapy for tachycardia and fibrillation[6]
» Calcium flux during calcium sparks in ventricular myocytes[7]
» Cardiac conduction as a function of cell rigidity in the context of cardiovascular disease[8]
» Diastolic Ca2+ transients in cardiac myocytes and SR-luminal and free cytoplasmic Ca2+ concentrations[9]
» Sphingosine-1-phosphate (S1P) receptor activation in valvular interstitial cells as detected by cytosolic Ca2+ flux[10]
In Neurobiology, Fluo-8® AM has been used to study:
» Hippocampal CA1 neurons, visualizating neurons to investigate the role of amyloid-β in the progression of Alzheimer's disease[11]
» Cytosolic Ca2+ concentrations in HEK293 cells and its regulatory effect on Aβ1-42 and hAmylin and associated signaling pathways [12]
» G protein-coupled receptors (GPRs) in response to cannabinoids in presynaptic CA3 or postsynaptic CA1 pyramidal cells [13]
» Medullary interneurons and dendritic calcium activity in regards to inspiratory bursts[14]
» N2a cell activation by histamine, as monitored by increases in intracellular Ca2+ concentrations[15]
In Stem Cells, Development & Differentiation, Fluo-8® AM has been used to study:
» Induction of pluripotent stem cells (iPSCs) into functioning cardiac cells, as validated by Ca2+ flux and membrane potential[16]
» CXCR4 and CXCR7 receptors in T cells and their role in cell survival and chemotaxis [17]
» Ca2+ uptake by myocytes derived from human induced pluripotent stem cells during pathogenesis of Duchenne muscular dystrophy[18]
» Agonist-induced calcium transients in differentiation of rat bone marrow mesenchymal stem cells into smooth muscle cells[19]
» Calcium channel blockades and their effect on cardiac progenitor cell proliferation and differentiation[20]
