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Calbryte™ 590 AM

An ATP dose-response was measured in CHO-K1 cells with Calbryte™ 590 AM. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 590 AM in HH Buffer with probenecid was added and incubated for 60 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
An ATP dose-response was measured in CHO-K1 cells with Calbryte™ 590 AM. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 590 AM in HH Buffer with probenecid was added and incubated for 60 min at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
Ordering information
Price ()
Catalog Number20700
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)1400
Molecular weight1218.77
SolventDMSO
Spectral properties
Excitation (nm)581
Emission (nm)593
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Molecular weight
1218.77
Dissociation constant (Kd, nM)
1400
Excitation (nm)
581
Emission (nm)
593
The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein"coupled receptors (GPCRs) and calcium channel targets. Followed by Rhod-2 being introduced in 1989, Rhod-4 and Cal-590 were later developed with improved signal/background ratio, and they became the widely used red fluorescent Ca2+ indicators for confocal microscopy, flow cytometry and high throughput screening applications. In CHO and HEK cells Rhod-4 and Cal-590 have cellular calcium response that are 10 times more sensitive than Rhod-2 AM. However, Cal-590 and Rhod-4 are still less sensitive to calcium in cells than the corresponding green fluorescent calcium indicators (e.g., Fluo-8 and Cal-520). Calbryte™ 590 is a new generation of red fluorescent indicators for the measurement of intracellular calcium. Its greatly improved signal/background ratio and intracellular retention properties make Calbryte™ 590 AM the most robust red fluorescent indicator for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists in live cells. Like other dye AM cell loading, Calbryte™ 590 AM ester is non-fluorescent and once gets inside the cell, it is hydrolyzed by intracellular esterase and gets activated. The activated indicator is a polar molecule that is no longer capable of freely diffusing through cell membrane, essentially trapped inside cells.

Platform


Fluorescence microscope

ExcitationTRITC/Cy3
EmissionTRITC/Cy3
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation540
Emission590
Cutoff570
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Calbryte™ 590 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calbryte™ 590 AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Calbryte™ 590 AM Working Solution
On the day of the experiment, either dissolve Calbryte™ 590 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Calbryte™ 590 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note     The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Calbryte™ 590 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note     If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solution, can be purchased from AAT Bioquest.

SAMPLE EXPERIMENTAL PROTOCOL

Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Calbryte™ 590 AM working solution into your cell plate.
    Note     If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
    Note     Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
  4. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
  5. Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a TRITC/Cy3 filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calbryte™ 590 AM to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM82.05 µL410.25 µL820.499 µL4.102 mL8.205 mL
5 mM16.41 µL82.05 µL164.1 µL820.499 µL1.641 mL
10 mM8.205 µL41.025 µL82.05 µL410.25 µL820.499 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)581
Emission (nm)593

Product family


NameExcitation (nm)Emission (nm)Quantum yield
Calbryte™ 520 AM4935150.751
Calbryte™ 630 AM607624-
Calbryte™-520L AM4935150.751
Calbryte™-520XL AM4935150.751
Cal-590™ AM5745880.621

Citations


View all 32 citations: Citation Explorer
Dissociation of inositol 1, 4, 5-trisphosphate from IP3 receptors contributes to termination of Ca2+ puffs
Authors: Smith, Holly A and Taylor, Colin W
Journal: Journal of Biological Chemistry (2023): 102871
The influence of spontaneous and visual activity on the development of direction selectivity maps in mouse retina
Authors: Tiriac, Alexandre and Bistrong, Karina and Pitcher, Miah N and Tworig, Joshua M and Feller, Marla B
Journal: Cell reports (2022): 110225
Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions
Authors: Cs{\'a}sz{\'a}r, Eszter and L{\'e}n{\'a}rt, Nikolett and Cser{\'e}p, Csaba and K{\"o}rnyei, Zsuzsanna and Fekete, Rebeka and P{\'o}sfai, Bal{\'a}zs and Bal{\'a}zsfi, Di{\'a}na and Hangya, Bal{\'a}zs and Schwarcz, Anett D and Szabadits, Eszter and others,
Journal: Journal of Experimental Medicine (2022)
Neurodegeneration in human brain organoids infected with herpes simplex virus type 1
Authors: Rybak-Wolf, Agnieszka and Wyler, Emanuel and Legnini, Ivano and Loewa, Anna and Gla{\v{z}}ar, Petar and Kim, Seung Joon and Pentimalli, Tancredi Massimo and Martinez, Anna Oliveras and Beyersdorf, Benjamin and Woehler, Andrew and others,
Journal: bioRxiv (2021)
KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals
Authors: Thillaiappan, Nagendra Babu and Smith, Holly A and Atakpa-Adaji, Peace and Taylor, Colin W
Journal: Nature communications (2021): 1--13
Evidence that polyphenols do not inhibit the phospholipid scramblase TMEM16F
Authors: Le, Trieu and Le, Son C and Zhang, Yang and Liang, Pengfei and Yang, Huanghe
Journal: Journal of Biological Chemistry (2020): 12537--12544
A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo
Authors: Dong, Ao and He, Kaikai and Dudok, Barna and Farrell, Jordan S and Guan, Wuqiang and Liput, Daniel J and Puhl, Henry L and Cai, Ruyi and Duan, Jiali and Albarran, Eddy and others,
Journal: BioRxiv (2020)
Ca2+ Release by IP3 Receptors Is Required to Orient the Mitotic Spindle
Authors: Lagos-Cabr{\'e}, Raul and Ivanova, Adelina and Taylor, Colin W
Journal: Cell reports (2020): 108483
TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development
Authors: Zhang, Yang and Le, Trieu and Grabau, Ryan and Mohseni, Zahra and Kim, Hoejeong and Natale, David R and Feng, Liping and Pan, Hua and Yang, Huanghe
Journal: Science advances (2020): eaba0310

References


View all 53 references: Citation Explorer
A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
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Journal: J Immunol Methods (2006): 220
Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy
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Journal: Gen Physiol Biophys (2004): 357
Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and microphysiometry
Authors: Patel H, Porter RH, Palmer AM, Croucher MJ.
Journal: Br J Pharmacol (2003): 671
Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics
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Journal: Cell Calcium (2003): 1
A sensitive method for the detection of foot and mouth disease virus by in situ hybridisation using biotin-labelled oligodeoxynucleotides and tyramide signal amplification
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Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) chang
Authors: Rockwell PL, Storey BT.
Journal: Mol Reprod Dev (2000): 335
MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells
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Journal: Am J Physiol Gastrointest Liver Physiol (2000): G522
Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue
Authors: Walczysko P, Wagner E, Albrechtova JT.
Journal: Cell Calcium (2000): 23
[Ca2+]i following extrasystoles in guinea-pig trabeculae microinjected with fluo-3 - a comparison with frog skeletal muscle fibres
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Journal: Acta Physiol Scand (2000): 1
Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations
Authors: Rockwell PL, Storey BT.
Journal: Mol Reprod Dev (1999): 418