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Calbryte™ 630 AM

Graph illustrates signal-to-noise (SNR) x 100%. ATP dose response was measured in CHO-K1 cells with Calbryte™ 630 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™ 630 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.
Graph illustrates signal-to-noise (SNR) x 100%. ATP dose response was measured in CHO-K1 cells with Calbryte™ 630 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™ 630 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.
The ATP induced intracellular calcium release was measured by Calbryte™ 630 AM. Cells were incubated with Calbryte™ 630 AM dye for 30 min at 37 °C before 10 µM ATP was added into the cells. The baseline was acquired and the rest of the cells were analyzed after the addition of ATP. The response was measured over time. The analysis was done on NovoCyte™ 3000 Flow Cytometer APC Channel.
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Price ()
Catalog Number20720
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)1200
Molecular weight1234.84
SolventDMSO
Spectral properties
Excitation (nm)607
Emission (nm)624
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
1234.84
Dissociation constant (Kd, nM)
1200
Excitation (nm)
607
Emission (nm)
624
Calcium measurement is critical for numerous biological investigations. Fluorescent probes 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. x-Rhod-1 is commonly used as a red fluorescent calcium indicator. However, x-Rhod-1 is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Calbryte™ 630 has been developed to improve x-Rhod-1 cell loading and calcium response while maintaining the spectral wavelength of x-Rhod-1, making it compatible with Texas Red® filter set. In CHO and HEK cells Cal-630™ AM has cellular calcium response that is much more sensitive than x-Rhod-1. The spectra of Calbryte™ 630 is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies. Calbryte™ 630 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™ 630 AM the most robust deep 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™ 630 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


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Fluorescence microscope

ExcitationTexas Red
EmissionTexas Red
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation600
Emission640
Cutoff630
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™ 630 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calbryte™ 630 AM in anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Calbryte™ 630 AM Working Solution
On the day of the experiment, either dissolve Calbryte™ 630 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™ 630 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™ 630 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™ 630 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 Texas Red filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 600/640 nm cutoff 630 nm. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Calbryte™ 630 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 mM80.982 µL404.911 µL809.822 µL4.049 mL8.098 mL
5 mM16.196 µL80.982 µL161.964 µL809.822 µL1.62 mL
10 mM8.098 µL40.491 µL80.982 µL404.911 µL809.822 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)607
Emission (nm)624

Citations


View all 21 citations: Citation Explorer
Using hiPSC-CMs to Examine Mechanisms of Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Arslanova, Alia and Shafaattalab, Sanam and Ye, Kevin and Asghari, Parisa and Lin, Lisa and Kim, BaRun and Roston, Thomas M and Hove-Madsen, Leif and Van Petegem, Filip and Sanatani, Shubhayan and others,
Journal: Current Protocols (2021): e320
RANK promotes colorectal cancer migration and invasion by activating the Ca 2+-calcineurin/NFATC1-ACP5 axis
Authors: Liang, Qian and Wang, Yun and Lu, Yingsi and Zhu, Qingqing and Xie, Wenlin and Tang, Nannan and Huang, Lifen and An, Tailai and Zhang, Di and Yan, Anqi and others,
Journal: Cell death \& disease (2021): 1--18
Calcium Channels Contributing to Action Potential Firing and Rhythms in the Circadian Clock
Authors: Plante, Amber E and Meredith, Andrea L
Journal: Biophysical Journal (2019): 240a
Null-Sarcolipin Equine Muscle Shows Enhanced SERCA Calcium Transport Which May Potentiate the Prevalence of Exertional Rhabdomyolysis
Authors: Autry, Joseph M and Svensson, Bengt and Karim, Christine B and Perumbakkam, Sudeep and Chen, Zhenhui and Finno, Carrie J and Thomas, David D and Valberg, Stephanie J
Journal: Biophysical Journal (2019): 238a--239a
The Arrhythmogenic E105A CAM Mutation Dysregulates Normal Cardiac Function in Zebrafish by Altering CAM-Ca2+ and CAM-RyR2 Interactions
Authors: Nomikos, Michail and Da'as, Sahar I and Thanassoulas, Angelos and Salem, Rola and Calver, Brian L and Saleh, Alaaeldin and Al-Maraghi, Ali and Nasrallah, Gheyath K and Safieh-Garabedian, Bared and Toft, Egon and others, undefined
Journal: Biophysical Journal (2019): 240a
Pade Approximation of Single-Channel Calcium Nanodomains in the Presence of Cooperative Calcium Buffers
Authors: Chen, Yinbo and Matveev, Victor
Journal: Biophysical Journal (2019): 239a--240a
LASS2 inhibits proliferation and induces apoptosis in HepG2 cells by affecting mitochondrial dynamics, the cell cycle and the nuclear factor-$\kappa$B pathways
Authors: Yang, Yan and Yang, Xiaoli and Li, Lin and Yang, Gangyi and Ouyang, Xuhong and Xiang, Jialin and Zhang, Tao and Min, Xun
Journal: Oncology reports (2019): 3005--3014
Ca2+ Diffusion in the Large Peptidergic Nerve Terminals of the Posterior Pituitary
Authors: McMahon, Shane M and Jackson, Meyer B
Journal: Biophysical Journal (2019): 238a
Functionally Identifying Members of the MscS Superfamily of Ion Channels in Paraburkholderia Membranes
Authors: Dickinson, Hannah M and Miller, Brittni L and Malcolm, Hannah R
Journal: Biophysical Journal (2019): 240a--241a
Shear stress induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells
Authors: Jetta, Deekshitha and Gottlieb, Philip A and Verma, Deepika and Sachs, Frederick and Hua, Susan Z
Journal: J Cell Sci (2019): jcs--226076

References


View all 53 references: Citation Explorer
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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
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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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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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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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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
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