Cal-590™ AM

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Additional ordering information

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

Physical properties

Dissociation constant (K_d, nM)	561
Molecular weight	1266.81
Solvent	DMSO

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	78000
Excitation (nm)	574
Emission (nm)	588
Quantum yield	0.62¹

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

Overview SDS Protocol

Molecular weight

1266.81

Dissociation constant (K_d, nM)

561

Extinction coefficient (cm ^-1 M ^-1)

78000

Excitation (nm)

574

Emission (nm)

588

Quantum yield

0.62¹

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. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Cal-590™ has been developed to improve Rhod-2 cell loading and calcium response while maintaining the spectral wavelength of Rhod-2, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells Cal-590™ AM has cellular calcium response that is much more sensitive than Rhod-2 AM. The spectra of Cal-590 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	TRITC/Cy3
Emission	TRITC/Cy3
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	540
Emission	590
Cutoff	570
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-590™ AM Stock Solution

Prepare a 2 to 5 mM stock solution of Cal-590™ AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Cal-590™ AM Working Solution

On the day of the experiment, either dissolve Cal-590™ 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-590™ 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-590™ 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-590™ 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 TRITC/Cy3 filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cal-590™ 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	78.938 µL	394.692 µL	789.384 µL	3.947 mL	7.894 mL
5 mM	15.788 µL	78.938 µL	157.877 µL	789.384 µL	1.579 mL
10 mM	7.894 µL	39.469 µL	78.938 µL	394.692 µL	789.384 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	78000
Excitation (nm)	574
Emission (nm)	588
Quantum yield	0.62¹

Product Family

Name	Excitation (nm)	Emission (nm)	Quantum yield
Cal-630™ AM	609	626	0.37¹
Cal-520®, AM	493	515	0.75¹
Cal-520FF™, AM	493	515	0.75¹
Cal-520N™, AM	493	515	0.75¹
Calbryte™ 590 AM	581	593	-
Cal-500™ AM	388	482	0.48¹

Images

Figure 1. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590® AM or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. 100 µL of 5 µg/mL Cal-590® AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.

Citations

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Glutamate drives ‘local Ca2+ release’in cardiac pacemaker cells
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Authors: Chen, Kevin H and Doliba, Nicolai and May, Catherine L and Roman, Jeffrey and Ustione, Alessandro and Tembo, Teguru and Negron, Ariel and Radovick, Sally and Piston, David W and Glaser, Benjamin and others,
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Gi/o protein--coupled receptor inhibition of beta--cell electrical excitability and insulin secretion depends on Na+/K+ ATPase activation
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Journal: bioRxiv (2022)

The Retinal Basis of Light Aversion in Neonatal Mice
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Journal: Journal of Neuroscience (2022): 4101--4115

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Authors: Huethorst, Eline
Journal: (2022)

Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit
Authors: Schulte, Annemarie and Bieniussa, Linda and Gupta, Rohini and Samtleben, Samira and Bischler, Thorsten and Doering, Kristina and Sodmann, Philipp and Rittner, Heike and Blum, Robert
Journal: Cell Calcium (2022): 102515

TMBIM5 loss of function alters mitochondrial matrix ion homeostasis and causes a skeletal myopathy
Authors: Zhang, Li and Dietsche, Felicia and Seitaj, Bruno and Rojas-Charry, Liliana and Latchman, Nadina and Tomar, Dhanendra and W{\"u}st, Rob CI and Nickel, Alexander and Frauenknecht, Katrin BM and Schoser, Benedikt and others,
Journal: Life science alliance (2022)

Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses
Authors: Jairaman, Amit and Othy, Shivashankar and Dynes, Joseph L and Yeromin, Andriy V and Zavala, Angel and Greenberg, Milton L and Nourse, Jamison L and Holt, Jesse R and Cahalan, Stuart M and Marangoni, Francesco and others,
Journal: Science advances (2021): eabg5859

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein
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Journal: Neurobiology of disease (2021): 105561