Cal-520FF™, 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)	9800
Molecular weight	1138.92
Solvent	DMSO

Spectral properties

Excitation (nm)	493
Emission (nm)	515
Quantum yield	0.75¹

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

1138.92

Dissociation constant (K_d, nM)

9800

Excitation (nm)

493

Emission (nm)

515

Quantum yield

0.75¹

Cal-520® provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cal-520® AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520® AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal-520™AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520®. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520® AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520® calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists. Compared to other Cal-520® indicators, Cal-520FF™ has the lowest affinity to calcium with Kd ~ 10 uM.

Platform

Fluorescence microscope

Excitation	FITC
Emission	FITC
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	490
Emission	525
Cutoff	515
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-520FF™ AM Stock Solution

Prepare a 2 to 5 mM stock solution of Cal-520FF™ AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Cal-520FF™ AM Working Solution

On the day of the experiment, either dissolve Cal-520FF™ 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-520FF™ 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-520FF™ 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-520FF™ 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 1 to 2 hours.
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 FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cal-520FF™, 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	87.802 µL	439.012 µL	878.025 µL	4.39 mL	8.78 mL
5 mM	17.56 µL	87.802 µL	175.605 µL	878.025 µL	1.756 mL
10 mM	8.78 µL	43.901 µL	87.802 µL	439.012 µL	878.025 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

Excitation (nm)	493
Emission (nm)	515
Quantum yield	0.75¹

Product Family

Name	Excitation (nm)	Emission (nm)	Quantum yield
Cal-520®, AM	493	515	0.75¹
Cal-520N™, AM	493	515	0.75¹
Cal-590™ AM	574	588	0.62¹
Cal-630™ AM	609	626	0.37¹
Cal-500™ AM	388	482	0.48¹

Citations

View all 95 citations: Citation Explorer

KS0365, a novel activator of the transient receptor potential vanilloid 3 (TRPV3) channel, accelerates keratinocyte migration
Authors: Maier, Marion and Olthoff, Stefan and Hill, Kerstin and Zosel, Carolin and Magauer, Thomas and Wein, Lukas Anton and Schaefer, Michael
Journal: British Journal of Pharmacology (2022): 5290--5304

Cal-520FF is the Present Optimal Ca 2+ Indicator for Ultrafast Ca 2+ Imaging and Optical Measurement of Ca 2+ Currents
Authors: Bl{\"o}mer, Laila Ananda and Filipis, Luiza and Canepari, Marco
Journal: Journal of Fluorescence (2021): 619--623

Advances in Two-Photon Scanning and Scanless Microscopy Technologies for Functional Neural Circuit Imaging
Authors: Schultz, Simon R and Copel, undefined and , Caroline S and Foust, Am and a J , undefined and Quicke, Peter and Schuck, Renaud
Journal: Proceedings of the IEEE (2017): 139--157

Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators
Authors: Hashitani, Hikaru and Nguyen, Michael J and Noda, Haruka and Mitsui, Retsu and Higashi, Ryuhei and Ohta, Keisuke and Nakamura, Kei-Ichiro and Lang, Richard J
Journal: Pflügers Archiv-European Journal of Physiology (2017): 1--17

Extensive Ca 2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca 2+ hypersensitivity
Authors: Uehara, Akira and Murayama, Takashi and Yasukochi, Midori and Fill, Michael and Horie, Minoru and Okamoto, Toru and Matsuura, Yoshiharu and Uehara, Kiyoko and Fujimoto, Takahiro and Sakurai, Takashi and others, undefined
Journal: The Journal of general physiology (2017): 199--218

Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy Complex Spike Synchronicity during GSWDs
Authors: Kros, Lieke and Lindeman, S and er , undefined and Eelkman Rooda, Oscar HJ and Murugesan, Pavithra and Bina, Lorenzo and Bosman, Laurens WJ and De Zeeuw, Chris I and Hoebeek, Freek E
Journal: Frontiers in Cellular Neuroscience (2017): 346

Simultaneous Measurement of Neural Activities of Acute Mouse Hippocampal Slices Using Multi-Electrode Array System and Laser Confocal Calcium Imaging
Authors: Hamasaki, Yuuta and Haba, Natsumi and Iwata, Naoki and Uno, Yoshiki and Saito, Minoru
Journal: Journal of Behavioral and Brain Science (2017): 68

Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions
Authors: Thillaiappan, Nagendra Babu and Chavda, Alap P and Tovey, Stephen C and Prole, David L and Taylor, Colin W
Journal: Nature Communications (2017): 1505

Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone
Authors: Yu, Kanglun and Sellman, David P and Bahraini, Anoosh and Hagan, Mackenzie L and Elsherbini, Ahmed and Vanpelt, Kayce T and Marshall, Peyton L and Hamrick, Mark W and McNeil, Anna and McNeil, Paul L and others, undefined
Journal: Journal of Orthopaedic Research (2017)

Neonatal CX26 removal impairs neocortical development and leads to elevated anxiety
Authors: Su, Xin and Chen, Jing-Jing and Liu, Lin-Yun and Huang, Qian and Zhang, Li-Zhao and Li, Xiao-Yang and He, Xiang-Nan and Lu, Wenlian and Sun, Shan and Li, Huawei and others, undefined
Journal: Proceedings of the National Academy of Sciences (2017): 201613237