Cal-630™ AM
Ordering information
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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) | 792 |
Molecular weight | 1282.89 |
Solvent | DMSO |
Spectral properties
Excitation (nm) | 609 |
Emission (nm) | 626 |
Quantum yield | 0.371 |
Storage, safety and handling
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 | ![]() ![]() |
Molecular weight 1282.89 | Dissociation constant (Kd, nM) 792 | Excitation (nm) 609 | Emission (nm) 626 | Quantum yield 0.371 |
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. Cal-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 Cal-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.
Platform
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.
Cal-630™ AM Stock Solution
Prepare a 2 to 5 mM stock solution of Cal-630™ AM in high-quality, anhydrous DMSO.PREPARATION OF WORKING SOLUTION
Cal-630™ AM Working Solution
On the day of the experiment, either dissolve Cal-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, Cal-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 Cal-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 Cal-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 2 hours 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 Cal-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 | 77.949 µL | 389.745 µL | 779.49 µL | 3.897 mL | 7.795 mL |
5 mM | 15.59 µL | 77.949 µL | 155.898 µL | 779.49 µL | 1.559 mL |
10 mM | 7.795 µL | 38.975 µL | 77.949 µL | 389.745 µL | 779.49 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Spectrum
Open in Advanced Spectrum Viewer


Spectral properties
Excitation (nm) | 609 |
Emission (nm) | 626 |
Quantum yield | 0.371 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-590™ AM | 574 | 588 | 0.621 |
Cal-520®, AM | 493 | 515 | 0.751 |
Cal-520FF™, AM | 493 | 515 | 0.751 |
Cal-520N™, AM | 493 | 515 | 0.751 |
Calbryte™ 630 AM | 607 | 624 | - |
Cal-500™ AM | 388 | 482 | 0.481 |
Images

Figure 1. ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-630™ AM (red curve). CHO-K1 cells were seeded overnight at 50,000 cells per 100 uL per well in a Costar black wall/clear bottom 96-well plate. 100 uL of 5 µM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. ATP (50 uL/well) was added using FlexSation to achieve the final indicated concentrations.

Figure 2. Response of endogenous P2Y receptor to ATP in CHO-K cells detected with Cal-630 ™ AM. 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 uL of 5 uM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. Images were recorded with a fluorescence microscope (Olympus IX71) before and after adding 10 uM ATP (final in the well) using Texas Red Channel.
Citations
View all 10 citations: Citation Explorer
Triple-Decker Sandwich Cultures of Intestinal Organoids for Long-Term Live Imaging, Uniform Perturbation, and Statistical Sampling
Authors: Cambra, Hailey M and Tallapragada, Naren P and Mannam, Prabhath and Breault, David T and Klein, Allon M
Journal: Current protocols (2022): e330
Authors: Cambra, Hailey M and Tallapragada, Naren P and Mannam, Prabhath and Breault, David T and Klein, Allon M
Journal: Current protocols (2022): e330
Tetraspanin-7 regulation of L-type voltage-dependent calcium channels controls pancreatic $\beta$-cell insulin secretion
Authors: Dickerson, Matthew T and Dadi, Prasanna K and Butterworth, Regan B and Nakhe, Arya Y and Graff, Sarah M and Zaborska, Karolina E and Schaub, Charles M and Jacobson, David A
Journal: The Journal of Physiology (2020): 4887--4905
Authors: Dickerson, Matthew T and Dadi, Prasanna K and Butterworth, Regan B and Nakhe, Arya Y and Graff, Sarah M and Zaborska, Karolina E and Schaub, Charles M and Jacobson, David A
Journal: The Journal of Physiology (2020): 4887--4905
Paracrine control of glucagon secretion in the pancreatic $\alpha$-cell: Studies involving optogenetic cell activation
Authors: Miranda, Caroline
Journal: (2020)
Authors: Miranda, Caroline
Journal: (2020)
Panaxadiol inhibits synaptic dysfunction in Alzheimer's disease and targets the Fyn protein in APP/PS1 mice and APP-SH-SY5Y cells
Authors: Liang, Xicai and Yao, Yingjia and Lin, Ying and Kong, Liang and Xiao, Honghe and Shi, Yue and Yang, Jingxian
Journal: Life Sciences (2019)
Authors: Liang, Xicai and Yao, Yingjia and Lin, Ying and Kong, Liang and Xiao, Honghe and Shi, Yue and Yang, Jingxian
Journal: Life Sciences (2019)
Development of a deep two-photon calcium imaging method for the analysis of cortical processing in the mammalian brain
Authors: Birkner, Antje
Journal: (2019)
Authors: Birkner, Antje
Journal: (2019)
Optical investigation of action potential and calcium handling maturation of hiPSC-cardiomyocytes on biomimetic substrates
Authors: Pioner, Jos{\`e} Manuel and Santini, Lorenzo and Palandri, Chiara and Martella, Daniele and Lupi, Flavia and Langione, Marianna and Querceto, Silvia and Grandinetti, Bruno and Balducci, Valentina and Benzoni, Patrizia and others,
Journal: International journal of molecular sciences (2019): 3799
Authors: Pioner, Jos{\`e} Manuel and Santini, Lorenzo and Palandri, Chiara and Martella, Daniele and Lupi, Flavia and Langione, Marianna and Querceto, Silvia and Grandinetti, Bruno and Balducci, Valentina and Benzoni, Patrizia and others,
Journal: International journal of molecular sciences (2019): 3799
Improvements in Simultaneous Sodium and Calcium Imaging
Authors: Miyazaki, Kenichi and Lisman, John E and Ross, William N
Journal: Frontiers in cellular neuroscience (2018)
Authors: Miyazaki, Kenichi and Lisman, John E and Ross, William N
Journal: Frontiers in cellular neuroscience (2018)
A circadian clock in the blood-brain barrier regulates xenobiotic efflux
Authors: Zhang, Shirley L and Yue, Zhifeng and Arnold, Denice M and Artiushin, Gregory and Sehgal, Amita
Journal: Cell (2018): 130--139
Authors: Zhang, Shirley L and Yue, Zhifeng and Arnold, Denice M and Artiushin, Gregory and Sehgal, Amita
Journal: Cell (2018): 130--139
A circadian clock in the blood-brain barrier regulates xenobiotic efflux from the brain
Authors: Zhang, Shirley L and Yue, Zhifeng and Arnold, Denice M and Sehgal, Amita
Journal: bioRxiv (2017): 196956
Authors: Zhang, Shirley L and Yue, Zhifeng and Arnold, Denice M and Sehgal, Amita
Journal: bioRxiv (2017): 196956
IP 3-Mediated Ca 2+ Signaling Deficit in Monogenic and Sporadic Forms of Autism Spectrum Disorders
Authors: Schmunk, Galina
Journal: (2017)
Authors: Schmunk, Galina
Journal: (2017)
References
View all 8 references: Citation Explorer
Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry
Authors: Stamm C, del Nido PJ.
Journal: Thorac Cardiovasc Surg (2004): 127
Authors: Stamm C, del Nido PJ.
Journal: Thorac Cardiovasc Surg (2004): 127
Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry
Authors: Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.
Journal: Cardiovasc Res (2003): 695
Authors: Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.
Journal: Cardiovasc Res (2003): 695
Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297
Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2
Authors: Del Nido PJ, Glynn P, Buenaventura P, Salama G, Koretsky AP.
Journal: Am J Physiol (1998): H728
Authors: Del Nido PJ, Glynn P, Buenaventura P, Salama G, Koretsky AP.
Journal: Am J Physiol (1998): H728
Application notes
A Meta-Analysis of Common Calcium Indicators
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calibration Protocol for Fluorescent Calcium Indicators
Novel Red Fluorescent Calcium Probes for Functional Analysis of GPCRs and Calcium Channel Targets
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
Calibration Protocol for Fluorescent Calcium Indicators
Novel Red Fluorescent Calcium Probes for Functional Analysis of GPCRs and Calcium Channel Targets
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
FAQ
How is intracellular calcium measured?
Are there any calcium indicators that don't require probenecid (PBC)?
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?
Are there any calcium indicators that don't require probenecid (PBC)?
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?