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AAT Bioquest

Calcium Indicators

Cell-based calcium flux assays are widely used in high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited signaling through G protein-coupled receptors (GPCRs), a large family of integral membrane proteins and an important target class in drug discovery. Accurate measurements of intracellular calcium concentration and mobilization allows for a more comprehensive understanding of calcium regulated signaling pathways, cellular functions and pathological processes.

AAT Bioquest has developed the largest collection of calcium indicators, including Fluo-8® , Cal-520®, and Calbryte™ calcium indicators, as well as, calcium flux assays to track calcium mobilization with intense fluorescent signals and in a variety of wavelength options ranging from ultraviolet to infrared.

 

 

Quantifying Cytosolic Calcium


Calcium is a ubiquitous messenger impacting a number of signal transduction pathways including calcium-induced changes in protein confirmation, the regulation of cytosolic and organelle calcium levels, the highly localized nature of calcium-mediated signal transduction and its specific roles in excitability, exocytosis, motility, apoptosis, and transcription. These pathways regulate many key cellular processes including neurotransmission, cell proliferation, and muscle contraction. Under normal conditions with respect to the extracellular fluid, the resting cytoplasmic concentration of calcium is relatively low, at or below 100 nM. Calcium signaling is activated either through the release of calcium ions from intracellular stores, or by an influx of extracellular calcium through plasma membrane ion channels. Stimulated cells can increase cytoplasmic calcium concentration up to 500 to 1000 nM.

Fluorescent AM Ester Derivatives for Live Cell Calcium Imaging


Fluorescent indicators that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. To facilitate detecting intracellular calcium mobilization and promote passive diffusion, acetoxymethyl ester (AM ester) functional groups are attached to the core structure of calcium indicators. This increases the indicator's hydrophobicity allowing it to easily penetrate intact plasma membranes, and enhances indicator sensitivity by minimizing non-specific background fluorescence because AM ester conjugated indicators are essentially non-fluorescent and inactive.

Activity is initiated once the probe enters a live cell and only after intracellular esterases remove the AM ester functional groups. Once activated, probes detect calcium through chelation and respond by generating a fluorescence signal relative to the concentration of calcium. Indicators such as Fluo-8®, AM (Cat No. 21080), Cal-520®, AM (Cat No. 21131) and Calbryte™ 520, AM (Cat No. 20653) have been extensively used in calcium imaging, calcium flux assays and cell-based high throughput screening (HTS) assays for drug discovery. Calcium indicators are compatible with use in flow cytometry, fluorescence microscopy, and the fluorescence microplate platform. Besides offering fluorescent and luminescent calcium indicators, we also provide several non-fluorescent calcium signaling molecules and chelators for measuring and manipulating intracellular and extracellular calcium.

 

Single-Wavelength Calcium Indicators


Single-wavelength calcium indicators are widely used to monitor calcium mobilization, image the spatial dynamics of calcium signaling, and in HTS cell-based pharmacological screening of agonist-stimulated and antagonist-inhibited signaling through GPCRs. These indicators exhibit significant calcium-dependent changes in fluorescence intensity at saturating calcium levels without shifting either their absorbance or emission wavelengths. We've developed a variety of novel calcium indicators, including the Fluo-8® series, the Cal-520® series and the Calbryte™ series, designed to trace calcium mobilization with the brightest fluorescent signals and a range of wavelength and affinity options to satisfy many applications.

Calcium GPCR Comparison

ATP response measured in CHO-K1 cells. 100 µL of 10 µg/ml Calbryte™ 520 AM (Cat No. 20653) in HH Buffer with probenecid or 10 µg/ml Fluo-4, AM (Cat No. 20550) in HH Buffer with probenecid was added and incubated for 45 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added to achieve the final indicated concentration of 10 µM and then imaged with microscope FITC channel (Keyence).


Key features of single-wavelength calcium indicators

  • Minimal background fluorescence at resting calcium levels
  • Improved signal-to-background ratios
  • Greatly enhanced cellular retention
  • Cell-permeant formats for live cell calcium analysis (compatible with imaging, flow cytometry and microplate assays, such as HTS)
  • Cell-impermeant formats for loading by microinjection, patch pipette, or pinocytic loading
  • Ultraviolet (UV), violet and visible light-excitable formats
  • Low-, medium- and high-affinity formats provides a sensitivity range to satisfy many applications
 

Table 1. Green Fluorescent Calcium Indicators for Intracellular Calcium Detection

Dye
Ex/Em (nm)
Filter Set
Kd¹
Φ²
FCa/FFree³
Relative Brightness
Signal/Noise
Unit Size
Cat No.
Calbryte™ 520, AM492/514FITC1.2 µM0.75∼300 fold**********10 x 50 µg20651
Cal-520®, AM492/514FITC320 nM0.75∼100 fold******10 x 50 µg21130
Fluo-8®, AM494/515FITC389 nM0.16∼200 fold******10 x 50 µg21082
Fluo-4, AM492/527FITC345 nM0.16∼100 fold****10 x 50 µg20551
Fluo-3, AM506/515FITC390 nM0.15∼100 fold**20 x 50 µg21013
Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]498/517FITC190 nM0.75∼14 fold******10 x 50 µg20501
Cal Green™ 1, hexapotassium salt498/517FITC190 nM0.75∼14 fold******10 x 50 µg20500
  1. Kd = Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
  3. FCa/FFree = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Table 2. Long Wavelength Rhod Calcium Indicators for Intracellular Calcium Detection

Dye
Ex/Em (nm)
Filter Set
Kd¹
Unit Size
Cat No.
Rhod-2, AM552/576TRITC570 nM20 x 50 µg21064
Rhod-4, AM521/549TRITC451 nM10 x 50 µg21122
Rhod-5N, AM557/579TRITC300 µM1 mg21070
Rhod-FF, AM552/576TRITC19 µM10 x 50 µg21078
  1. Kd = Calcium dissociation constant.

 

Ratiometric Calcium Indicators


Fura8vsFura2
ATP Dose response in CHO-K1 cells measured with Fura-2 AM (Cat No. 21023) and Fura-8™ AM (Cat No. 21056). CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate and then incubated with either Fura-2 AM or Fura-8 AM calcium assay dye-loading solution for 1 hour. ATP Dose was added by Flexstation.
In comparison to single-wavelength indicators, ratiometric (or dual-wavelength) indicators possess unique spectral properties which are triggered in response to binding calcium. When binding to calcium, ratiometric indicators undergo a shift in either their optimum absorbance or emission wavelength intensities. For example, dual-excitation calcium indicators Fura-2, AM and Fura-8™, AM exhibit two peak excitation wavelengths when either bound or free of calcium. An increase in calcium concentration initiates an increase and decrease in the fluorescence emission intensities of the dual-excitation indicator when excited at the calcium-bound and calcium-free peak excitation wavelengths, respectively.

Key features of ratiometric calcium indicators

  • Ratiometric determination produces accurate measurement of intracellular calcium concentrations
  • Minimizes effects of uneven dye loading, dye leakage, photobleaching and varying cell thickness (common in mixed populations)
  • Delivers more robust and reproducible results
  • Indicators available in dual-excitation, dual-emission or dual-excitation and dual-emission formats
  • Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
  • Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading

Learn more

 

Table 3. Dual-excitation ratiometric calcium indicators for live cell calcium imaging.

Dye
Ex/Em (zero calcium)
Ex/Em (high calcium)
Filter Set
Kd¹
Unit Size
Cat No.
Fura-2, AM *UltraPure Grade* *CAS 108964-32-5*340/510380/510Fura 2145 nM1 mg21021
Fura-2, AM *UltraPure Grade* *CAS 108964-32-5*340/510380/510Fura 2145 nM20x50 µg21023
Fura-2, AM *CAS 108964-32-5*340/510380/510Fura 2145 nM1 mg21020
Fura-2, AM *CAS 108964-32-5*340/510380/510Fura 2145 nM50 mg21022
Fura-2, pentapotassium salt *CAS 113694-64-7*340/510380/510Fura 2145 nM1 mg21025
Fura-2, pentasodium salt340/510380/510Fura 2145 nM1 mg21026
Fura-FF, AM *CAS 348079-12-9*340/510380/510Fura 25.5 µM10x50 µg21027
Fura-FF, pentapotassium salt *CAS 192140-58-2*340/510380/510Fura 25.5 µM10x50 µg21028
Fura-FF, pentapotassium salt *CAS 192140-58-2*340/510380/510Fura 25.5 µM1 mg21029
Fura-8™, AM355/525415/525Fura 2260 nM10x50 µg21056
Fura-8™, AM355/525415/525Fura 2260 nM1 mg21055
Fura-8™, pentapotassium salt355/525415/525Fura 2260 nM1 mg21057
Fura-8™, pentasodium salt355/525415/525Fura 2260 nM1 mg21058
Fura-8FF™, AM355/525415/525Fura 26 µM10x50 µg20620
Fura-8FF™, pentapotassium salt355/525415/525Fura 26 µM10x50 µg20621
Fura-10™, AM355/525415/525Fura 2N/D5x50 µg21114
Fura-10™, AM355/525415/525Fura 2N/D1 mg21115
Fura-10™, potassium salt355/525415/525Fura 2N/D5x50 µg21110
Fura-10™, potassium salt355/525415/525Fura 2N/D1 mg21111
  1. Kd = Calcium dissociation constant.

Table 4. Dual-emission ratiometric calcium indicators for live cell calcium imaging.

Dye
Ex/Em (zero calcium)
Ex/Em (high calcium)
Filter Set
Kd¹
Unit Size
Cat No.
Indo-1, AM *CAS 112926-02-0*355/475355/400Indo-1230 nM1 mg21030
Indo-1, AM *CAS 112926-02-0*355/475355/400Indo-1230 nM50 mg21033
Indo-1, AM *UltraPure Grade* *CAS 112926-02-0*355/475355/400Indo-1230 nM1 mg21032
Indo-1, AM *UltraPure Grade* *CAS 112926-02-0*355/475355/400Indo-1230 nM20x50 µg21036
Indo-1, pentapotassium salt *CAS 132319-21-9*355/475355/400Indo-1230 nM1 mg21040
Indo-1, pentasodium salt355/475355/400Indo-1230 nM1 mg21044
Cal Red™ R525/650 AM490/660490/525FITC230 nM10x50 µg20591
Cal Red™ R525/650 AM490/660490/525FITC230 nM1 mg20590
Cal Red™ R525/650 potassium salt490/660490/525FITC230 nM5x50 µg20588
  1. Kd = Calcium dissociation constant.

Table 5. Dual-excitation and dual-emission ratiometric calcium indicators for live cell calcium imaging.

Dye
Ex/Em (zero calcium)
Ex/Em (high calcium)
Cutoff
Kd¹
Unit Size
Cat No.
Fura Red, AM *CAS 149732-62-7*435/630470/650610, 630400 nM10x50 µg21048
Fura Red, AM *CAS 149732-62-7*435/630470/650610, 630400 nM1 mg21046
Fura Red, potassium salt435/630470/650610, 630400 nM10x50 µg21047
Fura Red, potassium salt435/630470/650610, 630400 nM1 mg21045
  1. Kd = Calcium dissociation constant.

 

Calbryte™ Calcium Indicators


The Calbryte™ series is a family of the brightest and most robust calcium indicators developed to monitor intracellular calcium mobilization. It includes three novel calcium indicators, Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630, which differ primarily in their absorbance and emission wavelengths. Calbryte™ probes emit fluorescence signals at significantly higher signal-to-background characteristics, and have greatly enhanced cellular retention. The Calbryte™ series has been optimized for use with fluorescence microscopes, fluorescence microplate readers and flow cytometers. They can also be used in high-throughput screening applications.

ATP-stimulated calcium response

Probenecid-free ATP -induced calcium response measured in CHO-K1 cells. 100 µL of 10 µg/mL Calbryte-520 AM in HH Buffer or 10 µg/mL Fluo-4 in HH Buffer was added and incubated for 45 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. Carbachol (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.


Key features of Calbryte™ calcium indicators

  • Proven performance, superior signal-to-noise ratio and intracellular retention properties
  • Calbryte™ probes are significantly brighter than any other calcium indicators under the same conditions
  • Greatly improved signal-to-background ratios
  • Enhanced intracellular retention, eliminates the use of probenecid
  • Fast and convenient cell loading in 45 to 60 minutes
  • Homogenous cytosolic localization
  • Red-shifted Calbryte™ 590 and Calbryte™ 630 can be used with GFP or other green-fluorescent dyes for multiplexing
  • Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
  • Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading
  • Available as a reactive-dye for conjugation to a carrier
 

Table 6. Calbryte™ calcium indicators for live cell calcium imaging.

Dye
Ex (nm)
Em (nm)
Filter Set
Kd¹
FCa/FFree²
Unit Size
Cat No.
Calbryte™ 520, AM493515FITC1.2 µM∼300 fold2x50 µg20650
Calbryte™ 520, AM493515FITC1.2 µM∼300 fold10x50 µg20651
Calbryte™ 520, AM493515FITC1.2 µM∼300 fold1 mg20653
Calbryte™ 520, potassium salt493515FITC1.2 µM∼300 fold2x50 µg20656
Calbryte™ 520, potassium salt493515FITC1.2 µM∼300 fold10x50 µg20658
Calbryte™ 520L, AM493515FITC100 µM∼300 fold10x50 µg20640
Calbryte™ 520L, potassium salt493515FITC100 µM∼300 fold10x50 µg20642
Calbryte™-520XL azide493515FITC300 µM∼300 fold1 mg20643
Calbryte™-520XL azide493515FITC300 µM∼300 fold10x50 µg20645
Calbryte™ 590, AM581593Cy3/TRITC1.4 µM∼300 fold2x50 µg20700
Calbryte™ 590, AM581593Cy3/TRITC1.4 µM∼300 fold10x50 µg20701
Calbryte™ 590, AM581593Cy3/TRITC1.4 µM∼300 fold1 mg20702
Calbryte™ 590, potassium salt581593Cy3/TRITC1.4 µM∼300 fold5x50 µg20706
Calbryte™ 590, potassium salt581593Cy3/TRITC1.4 µM∼300 fold1 mg20707
Calbryte™ 630, AM607624Texas Red1.2 µM∼300 fold2x50 µg20720
Calbryte™ 630, AM607624Texas Red1.2 µM∼300 fold10x50 µg20721
Calbryte™ 630, AM607624Texas Red1.2 µM∼300 fold1 mg20722
Calbryte™ 630, potassium salt607624Texas Red1.2 µM∼300 fold5x50 µg20727
  1. Kd = Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
  3. FCa/FFree = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

 

Cal-520® Calcium Indicators


Cal520vsFluo4
ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520® AM (red curve), or Fluo-4 AM (blue curve) respectively without probenecid. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520® AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37°C for 1 hour. ATP (50 µL/well) was added using FlexSation to achieve the final indicated concentrations.
The Cal-520® calcium indicators series is the most comprehensive portfolio of fluorogenic calcium-sensitive dyes. It includes six unique indicators, Cal-500™, Cal-520®, Cal-590™, Cal-630™, Cal-670™ and Cal-770™, which differ primarily in their absorbance and emission wavelengths. Each of the indicators emit minimal fluorescence at resting levels of calcium, and increases its fluorescence intensity >100-fold at saturating calcium concentrations. The Cal-520® series of indicators are available as membrane-permeant AM esters, and membrane-impermeant salt derivatives and dextran conjugates.

Key features of Cal-520® calcium indicators

  • Higher signal-to-background ratios than Fluo-3, Fluo-4, Rhod-2 and Rhod-5
  • Enhanced intracellular retention
  • Convenient and efficient cell loading
  • Homogenous cytosolic localization
  • Red-shifted Cal-590™, Cal-630™, Cal-670™ and Cal-770™ can be used with GFP or other green-fluorescent dyes for multiplexing
  • Cal-770™ may be used for in vivo studies
  • Available as membrane-permeant AM esters (compatible with imaging, flow cytometry and microplate assays, such as HTS)
  • Available as membrane-impermeant salt derivatives for loading by microinjection, patch pipette, or pinocytic loading
  • Available as reactive-dyes for conjugation to a carrier
 

Table 7. Cal-520® calcium indicators for live cell calcium imaging and multicolor calcium flux assays.

Dye
Ex (nm)
Em (nm)
Filter Set
Kd¹
Φ²
FCa/FFree³
Unit Size
Cat No.
Cal-500™, AM388482DAPI303 nM0.48∼100 fold10x50 µg20412
Cal-500™, potassium salt388482DAPI303 nM0.48∼100 fold10x50 µg20410
Cal-520®, AM493515FITC320 nM0.75∼100 fold10x50 µg21130
Cal-520®, potassium salt493515FITC320 nM0.75∼100 fold10x50 µg21140
Cal-590™ AM574588Cy3/TRITC561 nM0.62∼100 fold5x50 µg20510
Cal-590™ potassium salt574588Cy3/TRITC561 nM0.62∼100 fold5x50 µg20518
Cal-630™ AM609626Texas Red792 nM0.37∼100 fold5x50 µg20530
Cal-630™, potassium salt609626Texas Red792 nM0.37∼100 fold5x50 µg20538
Cal-670™, potassium salt667680Cy5853 nMN/D∼100 fold10x50 µg20455
Cal-770™, potassium salt758783Cy7850 nMN/D∼100 fold10x50 µg20460
Cal-520®, AM493515FITC320 nM0.75∼100 fold1 mg21131
Cal-520FF™, AM493515FITC9.8 µM0.75∼100 fold10x50 µg21143
Cal-520FF™, AM493515FITC9.8 µM0.75∼100 fold1 mg21142
Cal-520N™, AM493515FITC90 µM0.75∼100 fold10x50 µg21146
Cal-520®, potassium salt493515FITC320 nM0.75∼100 fold1 mg21141
Cal-520®, sodium salt493515FITC320 nM0.75∼100 fold10x50 µg21135
Cal-520®, sodium salt493515FITC320 nM0.75∼100 fold1 mg21136
Cal-520FF™, potassium salt493515FITC9.8 µM0.75∼100 fold10x50 µg21144
Cal-520N™, potassium salt493515FITC90 µM0.75∼100 fold10x50 µg21147
Cal-520®-Dextran Conjugate *MW 3,000*493515FITC320 nM0.75∼100 fold1 mg20600
Cal-520®-Dextran Conjugate *MW 10,000*493515FITC320 nM0.75∼100 fold5 mg20601
Cal-520L®-Dextran Conjugate *MW 10,000*493515FITC 0.75∼100 fold1 mg20602
Cal-520®-Biotin Conjugate493515FITC320 nM0.75∼100 fold5x50 µg20605
Cal-520®-Biocytin Conjugate493515FITC320 nM0.75∼100 fold5x50 µg20606
Cal-520® NHS Ester493515FITC320 nM0.75∼100 fold100 µg20609
Cal-520® maleimide493515FITC320 nM0.75∼100 fold100 µg20610
Cal-520L™ maleimide493515FITC 0.75∼100 fold100 ug20611
Cal-590™ AM574588Cy3/TRITC561 nM0.62∼100 fold10x50 µg20511
Cal-590™ AM574588Cy3/TRITC561 nM0.62∼100 fold1 mg20512
Cal-590™ potassium salt574588Cy3/TRITC561 nM0.62∼100 fold1 mg20519
Cal-590™ sodium salt574588Cy3/TRITC561 nM0.62∼100 fold5x50 µg20515
Cal-590™-Dextran Conjugate *MW 3,000*574588Cy3/TRITC561 nM0.62∼100 fold1 mg20508
Cal-590™-Dextran Conjugate *MW 10,000*574588Cy3/TRITC561 nM0.62∼100 fold1 mg20509
Cal-590L® Dextran Conjugate *MW 10,000*569590Cy3/TRITC∼230 uM ∼100 fold1 mg20525
Cal-630™ AM609626Texas Red792 nM0.37∼100 fold10x50 µg20531
Cal-630™ AM609626Texas Red792 nM0.37∼100 fold1 mg20532
Cal-630™, sodium salt609626Texas Red792 nM0.37∼100 fold5x50 µg20535
Cal-630™-Dextran Conjugate *MW 10,000*609626Texas Red792 nM0.37∼100 fold1 mg20546
Cal-670™-Dextran Conjugate *MW 3,000*667680Cy5853 nMN/D∼100 fold1 mg20456
Cal-670™-Dextran Conjugate *MW 10,000*667680Cy5853 nMN/D∼100 fold1 mg20457
Cal-770™-Dextran Conjugate *MW 3,000*758783Cy7853 nMN/D∼100 fold1 mg20461
Cal-770™-Dextran Conjugate *MW 10,000*758783Cy7853 nMN/D∼100 fold1 mg20462
  1. Kd = Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
  3. FCa/FFree = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.
  4. N/D = not determined.

 

Fluo 8® Calcium Indicators


Carbachol-stimulated calcium response
Carbachol dose response measured in HEK-293 cells. Hek-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye loading solution using Fluo-8® AM (Cat No. 21083) or Fluo-4 AM (Cat No. 20550) for 1 hour at room temperature. Carbachol (50 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
Fluo-8® AM addresses one of the major limitations common to fluo-based dyes, namely, the harsh conditions required for cell loading. Traditional fluo-based dyes such as Fluo- 2 AM (Cat No. 20495), Fluo-3 AM (Cat No. 21013 and Fluo-4 AM (Cat No. 20550) need to be loaded and incubated with cells at 37°C for up to an hour before a reading can be taken. This not only subjects cells to harsh temperatures, but also requires users to spend time heating and cooling cell cultures. Additionally, it introduces incubation temperature as a possible confounding variable. Fluo-8® AM resolves all these problem by providing a robust calcium indicator that can be loaded into cells at room temperature. Fluo-8® AM is less temperature dependent than other fluo-based dyes, allowing for more consistent results under a wider range of experimental conditions. Furthermore, with an incubation time of as little as 20 minutes, Fluo-8® AM enables fast turnaround for improved productivity. The best part is that even with structural modifications and improvements, Fluo-8® AM's spectral properties remains unchanged, maintaining the familiar spectra of Fluo-4 AM (ie. Ex/Em = 494/517nm).

Key features of Fluo 8® calcium indicators

  • Faster cell loading: Fluo 8® can be readily loaded into cells at room temperature. Fluo-3 AM and Fluo-4 AM require a loading temperature of 37°C
  • Brighter fluorescence: Fluo 8® is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM
  • Convenient: Fluo 8®'s identical spectra to those of Fluo-4 and Fluo-3 makes upgrading easy

Learn more

 

Table 8. Fluo-8® green fluorescent calcium indicators for live cell calcium imaging.

Indicator
Ex (nm)
Em (nm)
Kd¹
Φ²
FCa/FFree³
Unit Size
Cat No.
Fluo-8®, AM495516389 nM0.16∼200 fold1 mg21080
Fluo-8®, AM495516389 nM0.16∼200 fold10x50 µg21082
Fluo-8®, AM495516389 nM0.16∼200 fold20x50 µg21083
Fluo-8®, AM495516389 nM0.16∼200 fold5x50 µg21081
Fluo-8H™, AM495516232 nM0.16∼200 fold10x50 µg21091
Fluo-8H™, AM495516232 nM0.16∼200 fold1 mg21090
Fluo-8L™, AM4955161.9 µM0.16∼200 fold1 mg21096
Fluo-8L™, AM4955161.9 µM0.16∼200 fold10x50 µg21097
Fluo-8FF™, AM49551610 µM0.16∼200 fold10x50 µg21104
Fluo-8FF™, AM49551610 µM0.16∼200 fold1 mg21105
Fluo-8®, potassium salt495516389 nM0.16∼200 fold1 mg21087
Fluo-8®, potassium salt495516389 nM0.16∼200 fold10x50 µg21089
Fluo-8®, sodium salt495516389 nM0.16∼200 fold1 mg21086
Fluo-8®, sodium salt495516389 nM0.16∼200 fold10x50 µg21088
Fluo-8H™, sodium salt495516232 nM0.16∼200 fold10x50 µg21095
Fluo-8L™, sodium salt4955161.9 µM0.16∼200 fold10x50 µg21098
Fluo-8L™, sodium salt4955161.9 µM0.16∼200 fold1 mg21099
  1. Kd = Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
  3. FCa/FFree = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

 

Fluo-3 and Fluo-4 Calcium Indicators


Fluo-3 is a calcium indicator first introduced in 1989 for use in flow cytometry and confocal laser-scanning microscopy. A fluorescein-like compound, Fluo-3 is effectively non-fluorescent in the absence of free Ca2+. Upon binding to calcium, however, Fluo-3 experiences a sharp increase in emission intensity at 525 nm. As an indicator, Fluo-3 has been essential in the study of cell signaling pathways, myocytes and the activation of neurotransmitters.

Fluo-4 builds upon Fluo-3's core structure, but modifying it for increased fluorescence signal strength. The modification in chemical structure causes a blue-shift in Fluo-4's excitation maximum, from 506nm (of Fluo-3) to 494nm. Practically speaking, this 12 nm shift moves Fluo-4's excitation maximum closer to the wavelength of a standard argon laser (ie. 488nm). This results in better excitation and thus, a fluorescent signal that is significantly brighter and more sensitive to changes in calcium concentration.

Fluo 3, AM
Fluo3
Fluo 4, AM
Fluo4
Fluo 8, AM
Fluo8

U2OS 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 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 µM in a 37°C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

 

Table 9. Fluo-3, Fluo-4 and Fluo-5 green fluorescent calcium indicators for live cell calcium imaging.

Indicator
Ex (nm)
Em (nm)
Filter Set
Kd¹
Φ²
FCa/FFree³
Unit Size
Cat No.
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*507516FITC390 nM0.15∼100 fold1 mg21011
Fluo-3, AM *Bulk package* *CAS 121714-22-5*507516FITC390 nM0.15∼100 fold50 mg21012
Fluo-3, AM *CAS 121714-22-5*507516FITC390 nM0.15∼100 fold1 mg21010
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*507516FITC390 nM0.15∼100 fold1 mg21013
Fluo-3, pentapotassium salt507516FITC390 nM0.15∼100 fold1 mg21017
Fluo-3, pentaammonium salt507516FITC390 nM0.15∼100 fold1 mg21018
Fluo-3, pentasodium salt507516FITC390 nM0.15∼100 fold1 mg21016
Fluo-3FF, pentapotassium salt507516FITC100 µM0.15∼100 fold1 mg21019
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528FITC345 nM0.16∼100 fold1 mg20550
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528FITC345 nM0.16∼100 fold10x50 µg20551
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528FITC345 nM0.16∼100 fold5x50 µg20552
Fluo-4, Pentapotassium Salt495528FITC345 nM0.16∼100 fold1 mg20555
Fluo-4, Pentapotassium Salt495528FITC345 nM0.16∼100 fold1 mg20556
Fluo-5F, AM494516FITC2.3 µM0.16∼100 fold10x50 µg20560
Fluo-5N, AM494516FITC90 µM0.16∼100 fold10x50 µg20566
Fluo-5F, pentapotassium salt494516FITC2.3 µM0.16∼100 fold500 µg20562
Fluo-5N, pentapotassium salt494516FITC90 µM0.16∼100 fold500 µg20567
  1. Kd = Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in the presence of 5 mM calcium citrate.
  3. FCa/FFree = Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

 

Calcium Assays for Screening GPCR and Calcium Channel Targets


Carbachol-stimulated calcium response
Comparison of fluorescent signal response of endogenous P2Y receptor to ATP in CHO-K1 cells using Screen Quest™ Calbryte™ 520 Calcium Assay Kit, FLIPR® Calcium 4 Assay (Molecular Devices), and Fluo-4 Direct™ Calcium Assay (Molecular Probes®). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. Calcium flux response was measured with all three assays, respectively. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
Screen Quest™ calcium flux assays are the preferred method in cell-based high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited signaling through G protein coupled receptors (GPCRs). To date approximately one third of drugs available on the market are designed to target GPCRs. The Screen Quest™ Calcium Assay offers a convenient no-wash, homogeneous fluorescence-based assay for monitoring GPCRs and calcium channels, and to detect intracellular calcium mobilization across a broad spectrum of biological targets. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be easily adapted for automation. Screen Quest™ Calcium Assays are optimized and validated for the following instruments: FDSS®, FLIPR®, ViewLux™, NOVOStar, ArrayScan™, FlexStation® and IN Cell Analyzer.

Key Features of Screen Quest™ Calcium Assays

  • Optimized protocols validated for a broad range of microplate readers
  • Superior signal intensity (high S/B ratios) and well-to-well uniformity
  • Large assay window for assaying challenging cell lines and receptors
  • High performance calcium flux assays for 1536 well-plates
 

Table 10. Screen Quest™ Calcium Assays for screening GPCR and calcium channel targets.

Calcium Assay
Ex (nm)
Em (nm)
Cutoff (nm)
Unit Size
Cat No.
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit49052551510 plates36325
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit490525515100 plates36326
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit4905255101 plates36314
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit49052551010 plates36315
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit490525510100 plates36316
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit490525510100 plates36316
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*4905255101 plates36307
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*49052551010 plates36308
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*490525510100 plates36309
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit4905255151 plates36317
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit49052551510 plates36318
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit490525515100 plates36319
Screen Quest™ Fura-2 No Wash Calcium Assay Kit340, 38051047010 plates36320
Screen Quest™ Fura-2 No Wash Calcium Assay Kit340, 380510470100 plates36321
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit5405905701 plates36333
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit54059057010 plates36334
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit540590570100 plates36335
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*5405905701 plates36330
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*54059057010 plates36331
Screen Quest™ Rhod-4 No Wash Calcium Assay Kit *Medium Removal*540590570100 plates36332
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit5405905701 plates36200
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit54059057010 plates36201
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit540590570100 plates36202

 

GPCR Cell Lines for Drug Discovery and High Throughput Screening


Screen Quest™ cell lines are a series of cells that have been successfully used in drug discovery and screening environments for studying G-protein-coupled receptors (GPCR) that do not conventionally couple through intracellular calcium. It has been effectively used with the FLIPR, FDSS Systems in conjunction with non-Gq coupled members of many receptors such as chemokine, serotonin, glutamate, dopamine, opioid, vasopressin and α- and ß- adrenergic receptor families. Over 60% of the known GPCR signal through pathways other than Gq which lead to an increase in intracellular calcium, and as genomics reveals more G-protein-coupled receptor targets this trend continues to increase. Screen Quest™ cell lines are used for investigating GPCR that do not conventionally couple through intracellular calcium.
Screen Quest™ cell lines are based on a series of G-protein chimeras, including the promiscuous G-protein, Gα16. The chimeras consist of the alpha subunit of a Gq-protein complex whose 5 carboxy-terminal amino acids have been replaced with those from one of the other G-proteins (either Gαs, Gαi, Gαo, or Gαz). These amino acids are responsible for the coupling of the receptor to its G-protein. Co-expression of these chimeras with specific non-Gq-coupled receptors which normally act through the cAMP pathway may result in the generation of an intracellular calcium signal upon receptor stimulation.
 

Table 11. Stable GPCR cell lines for drug discovery and high-throughput screening assays.

Product name
Unit Size
Cat No.
Screen Quest™ HEK-CNGC-Amylin 3 Receptor CellsEach38000
Screen Quest™ HEK-CNGC-Cannabinoid Receptor 1 CellsEach38001
Screen Quest™ CHO-Ga16- Chemokine (C-C) Receptor 2B CellsEach38002
Screen Quest™ HEK-CNGC-Chemokine (C-X-C motif) Receptor 4 CellsEach38003
Screen Quest™ CHO-Ga16 Chimera Cell lineEach38105
Screen Quest™ CHO-Gqi Chimera Cell lineEach38101
Screen Quest™ CHO-Gqo Chimera Cell lineEach38102
Screen Quest™ CHO-Gqs Chimera Cell lineEach38104
Screen Quest™ CHO-Gqz Chimera Cell lineEach38103
Screen Quest™ HEK-CNGC-Dopamine Receptor 1 (DRD1) CellsEach38004
Screen Quest™ HEK-CNGC-Glucagon-like Receptor 1 (GLP1R) CellsEach38005
Screen Quest™ Human Nociceptin Receptor Ga16 coupled CHO Cells (NOP-Ga16)Each38100
Screen Quest™ Live Cell cAMP Assay Service PackEach36382
Screen Quest™ HEK-CNGC-Opiate Receptor-like 1 (ORL1) CellsEach38006

 

Ordering Information


 

Table 12. Cell Meter™ Calcium Assay Kits

Product Name
Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ Flow Cytometric Calcium Assay Kit493515100 Tests36310
Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*495525100 Tests36312

Table 13. Cell-Permeable Calcium Chelators

Product name
Unit Size
Cat No.
EDTA, AM ester10x50 µg19010
BAPTA, AM *CAS 126150-97-8*25 mg21001
BAPTA, AM *UltraPure Grade* *CAS 126150-97-8*25 mg21002
EGTA AM *CAS 99590-86-0*10 mg21005
EGTA AM *10 mM DMSO solution*1 mL21006

Table 14. Cell-Impermeable Calcium Chelators

Product name
Unit Size
Cat No.
BAPTA, tetrapotassium salt *CAS 73630-08-7*100 mg21003
BAPTA, tetrasodium salt *CAS 126824-24-6*100 mg21004
EGTA tetrasodium salt *CAS 13368-13-3*1 g21007
EGTA tetrasodium salt *10 mM aqueous solution*10 mL21008