Cal-630™ AM

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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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Dissociation constant (K_d, 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

Overview SDS Protocol

Molecular weight

1282.89

Dissociation constant (K_d, 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.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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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

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

Paracrine control of glucagon secretion in the pancreatic $\alpha$-cell: Studies involving optogenetic cell activation
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)

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