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Cal-630™ AM

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.
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.
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.
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.
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Physical properties
Dissociation constant (Kd, nM)792
Molecular weight1282.89
SolventDMSO
Spectral properties
Excitation (nm)609
Emission (nm)626
Quantum yield0.371
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
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

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

Cal-630™ AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Cal-630™ AM in anhydrous DMSO.

    Note: When reconstituted in DMSO, Cal-630™ AM is a clear, colorless solution.

PREPARATION OF WORKING SOLUTION

Cal-630™ AM Working Solution
  1. 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.

  2. Prepare a 2 to 20 µM Cal-630™ AM working solution 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 solutions, 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 Cal-630™ AM working solution to 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 2 hours 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 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 mg0.5 mg1 mg5 mg10 mg
1 mM77.949 µL389.745 µL779.49 µL3.897 mL7.795 mL
5 mM15.59 µL77.949 µL155.898 µL779.49 µL1.559 mL
10 mM7.795 µL38.975 µL77.949 µL389.745 µL779.49 µL

Molarity calculator

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

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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)609
Emission (nm)626
Quantum yield0.371

Product Family


NameExcitation (nm)Emission (nm)Quantum yield
Cal-590™ AM5745880.621
Cal-520®, AM4935150.751
Cal-520FF™, AM4935150.751
Cal-520N™, AM4935150.751
Calbryte™ 630 AM607624-
Cal-500™ AM3884820.481

Images


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
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
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)
Development of a deep two-photon calcium imaging method for the analysis of cortical processing in the mammalian brain
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
Improvements in Simultaneous Sodium and Calcium Imaging
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
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
IP 3-Mediated Ca 2+ Signaling Deficit in Monogenic and Sporadic Forms of Autism Spectrum Disorders
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
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
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
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
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
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
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
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