Calbryte™ 630 AM
Ordering information
| Price | |
| Catalog Number | |
| Unit Size | |
| Quantity |
Additional ordering information
| Telephone | 1-800-990-8053 |
| Fax | 1-800-609-2943 |
| sales@aatbio.com | |
| Quotation | Request |
| International | See distributors |
| Shipping | Standard overnight for United States, inquire for international |
Physical properties
| Dissociation constant (Kd, nM) | 1200 |
| Molecular weight | 1234.84 |
| Solvent | DMSO |
Spectral properties
| Excitation (nm) | 607 |
| Emission (nm) | 624 |
Storage, safety and handling
| Certificate of Origin | Download PDF |
| H-phrase | H303, H313, H333 |
| Hazard symbol | XN |
| Intended use | Research Use Only (RUO) |
| R-phrase | R20, R21, R22 |
| Storage | Freeze (< -15 °C); Minimize light exposure |
| UNSPSC | 12352200 |
Related products
| Overview |  SDSProtocol |
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
| Excitation | 640 nm laser |
| Emission | 660/20 nm filter |
| Instrument specification(s) | APC channel |
Fluorescence microscope
| Excitation | Texas Red |
| Emission | Texas Red |
| Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
| Excitation | 600 |
| Emission | 640 |
| Cutoff | 630 |
| Recommended plate | Black 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.
- Prepare cells in growth medium overnight.
- 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. - 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. - 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.
- 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 80.982 µL | 404.911 µL | 809.822 µL | 4.049 mL | 8.098 mL |
| 5 mM | 16.196 µL | 80.982 µL | 161.964 µL | 809.822 µL | 1.62 mL |
| 10 mM | 8.098 µL | 40.491 µL | 80.982 µL | 404.911 µL | 809.822 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Product Family
| Name | Excitation (nm) | Emission (nm) | Quantum yield |
| Calbryte™ 520 AM | 493 | 515 | 0.751 |
| Calbryte™ 590 AM | 581 | 593 | - |
| Calbryte™-520L AM | 493 | 515 | 0.751 |
| Calbryte™-520XL AM | 493 | 515 | 0.751 |
| Cal-630™ AM | 609 | 626 | 0.371 |
Images
Figure 1. 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.
Figure 2. 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.
Citations
View all 23 citations: Citation Explorer
Patient and cell-type specific hiPSC-modeling of a truncating titin variant associated with atrial fibrillation
Authors: Huang, Kate and Ashraf, Mishal and Rohani, Leili and Luo, Yinhan and Sacayanan, Ardin and Huang, Haojun and Haegert, Anne and Volik, Stanislav and Sar, Funda and LeBihan, St{\'e}phane and others,
Journal: bioRxiv (2023): 2023--03
Authors: Huang, Kate and Ashraf, Mishal and Rohani, Leili and Luo, Yinhan and Sacayanan, Ardin and Huang, Haojun and Haegert, Anne and Volik, Stanislav and Sar, Funda and LeBihan, St{\'e}phane and others,
Journal: bioRxiv (2023): 2023--03
Phenotypic screen identifies the natural product silymarin as a novel anti-inflammatory analgesic
Authors: DuBreuil, Daniel M and Lai, Xiaofan and Zhu, Kevin and Chahyadinata, Grace and Perner, Caroline and Chiang, Brenda and Battenberg, Ashley and Sokol, Caroline and Wainger, Brian
Journal: Molecular Pain (2022): 17448069221148351
Authors: DuBreuil, Daniel M and Lai, Xiaofan and Zhu, Kevin and Chahyadinata, Grace and Perner, Caroline and Chiang, Brenda and Battenberg, Ashley and Sokol, Caroline and Wainger, Brian
Journal: Molecular Pain (2022): 17448069221148351
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
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
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
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
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
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
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
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
Authors: McMahon, Shane M and Jackson, Meyer B
Journal: Biophysical Journal (2019): 238a
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
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
Journal: Gen Physiol Biophys (2004): 357
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
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
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
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
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
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187
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
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
Authors: Cantz T, Nies AT, Brom M, Hofmann AF, Keppler D.
Journal: Am J Physiol Gastrointest Liver Physiol (2000): G522
Authors: Cantz T, Nies AT, Brom M, Hofmann AF, Keppler D.
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.
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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
Authors: Wohlfart B., undefined
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Authors: Wohlfart B., undefined
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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
Authors: Rockwell PL, Storey BT.
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Authors: Rockwell PL, Storey BT.
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Application notes
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Calibration Protocol for Fluorescent Calcium Indicators
Introducing Calbryte™ Series
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
Calibration Protocol for Fluorescent Calcium Indicators
Introducing Calbryte™ Series
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
FAQ
Are there any calcium indicators that don't require probenecid (PBC)?
How to select a calcium indicator?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
How to select a calcium indicator?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?