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Cal Red™ R525/650 AM

Graph illustrates signal-to-noise ration (SNR) x 100%. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal Red R525/650. ATP (50 uL/well) was added by FlexStation3 (Molecular Devices) to achieve the final indicated concentrations.
Graph illustrates signal-to-noise ration (SNR) x 100%. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal Red R525/650. ATP (50 uL/well) was added by FlexStation3 (Molecular Devices) to achieve the final indicated concentrations.
Ordering information
Price ()
Catalog Number20590
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)330
Molecular weight~1100
SolventDMSO
Storage, safety and handling
Certificate of OriginDownload PDF
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
~1100
Dissociation constant (Kd, nM)
330
The intracellular calcium flux assay is a widely used method for monitoring the activities of GPCRs and calcium channels. To quantify the intracellular calcium concentration, ratiometric fluorescent calcium indicators are preferred because the ratio is directly related to the calcium concentration and independent of the cell numbers and dye loading concentration. However, the most popular ratiometric calcium indicators (such as Fura-2 and Indo-1) have certain limitations such as lower sensitivity, UV excitation, and not compatible with HTS screening filter set. Cal Red™ R525/650 has been developed as a new 488 nm-excitable ratiometric fluorescence calcium indicator. Cal Red™ R525/650 is weakly fluorescent, and once enters cells, the lipophilic AM blocking groups are cleaved by intracellular esterase, resulting in a negatively charged fluorescent dye retained well in cells with excitation close to 488 nm and two emissions at 525 nm and 650 nm. When cells are stimulated with a bioactive compound, the receptor initiates the release of intracellular calcium, which is chelated by Cal Red™ R525/650. The emission signal is increased at 525 nm and decreased at 650 nm when excited at 488 nm. The excitation and emission wavelength of Cal Red™ R525/650 are compatible with common filter sets with minimal damage to cells, making it a robust tool for evaluating and screening GPCR agonists and antagonists as well as calcium channel targets.

Platform


Fluorescence microscope

ExcitationFITC
EmissionFITC
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490
Emission525, 660
Cutoff515, 630
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 Red™ R525/650 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Cal Red™ R525/650 AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Cal Red™ R525/650 AM Working Solution
On the day of the experiment, either dissolve Cal Red™ R525/650 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 Red™ R525/650 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 Red™ R525/650 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.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Cal Red™ R525/650 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.
  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 1 hour 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 FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 490/525 nm cutoff 515 nm and Ex/Em2 = 490/660 nm cutoff 630 nm. 

Citations


View all 7 citations: Citation Explorer
A Portable Smartphone-Based System for the Detection of Blood Calcium Using Ratiometric Fluorescent Probes
Authors: Wu, Yue and Zhang, Yunshan and Xu, Zhongyuan and Guo, Xinyu and Yang, Wenjian and Zhang, Xiaoyu and Liao, Yuheng and Fan, Minzhi and Zhang, Diming
Journal: Biosensors (2022): 917
Characterization of Mitochondrial Calcium Uptake in Skeletal Muscle
Authors: Debattisti, Valentina and Paillard, Melanie and Csordas, Gyorgy and Seifert, Erin and Hajnoczky, Gyorgy
Journal: Biophysical Journal (2016): 259a
ER Calcium Levels Help Regulate K (ATP) Channel Trafficking to the Plasma Membrane of Pancreatic Beta Cells
Authors: Vadrevu, Suryakiran and Satin, Leslie S
Journal: Biophysical Journal (2016): 258a--259a
Interplay of Membrane and Calcium Oscillators in Cardiac Pacemaker Cells
Authors: Zorin, Nikolay and Ryvkin, Alexander and Moskvin, Alexander and Solovyova, Olga
Journal: Biophysical Journal (2016): 259a
A Novel Ratiometric Fluorescence Calcium Indicator for Functional Analysis of Gpcrs and Calcium Channel Targets
Authors: Diwu, Zhenjun and Zhao, Qin and Luo, Zhen and Meng, Qinglin and Liu, Jixiang and Liao, Jinfang
Journal: Biophysical Journal (2016): 259a
Receptor-Localized Ca2+ Signaling Activates P2X2 Receptor Changing Cytoskeletal Morphology
Authors: Qudrat, Anam and Truong, Kevin
Journal: Biophysical Journal (2016): 259a
Spontaneous, Local Diastolic Subsarcolemmal Ca2+ Releases (LCRS) in Single Isolated Guinea-PIG Sinoatrial Nodal Cells (SANC) are Linked to Their Spontaneous AP Firing
Authors: Sirenko, Syevda and Yang, Dongmei and Lakatta, Edward G
Journal: Biophysical Journal (2016): 259a--260a

References


View all 12 references: Citation Explorer
Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets
Authors: Wendt ER, Ferry H, Greaves DR, Keshav S.
Journal: PLoS One (2015): e0119532
A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue
Authors: Walczysko P, Wagner E, Albrechtova JT.
Journal: Cell Calcium (2000): 23
Monitoring calcium in outer hair cells with confocal microscopy and fluorescence ratios of fluo-3 and fura-red
Authors: Su ZL, Li N, Sun YR, Yang J, Wang IM, Jiang SC.
Journal: Shi Yan Sheng Wu Xue Bao (1998): 323
Problems associated with using Fura-2 to measure free intracellular calcium concentrations in human red blood cells
Authors: Blackwood AM, Sagnella GA, Mark and u ND, MacGregor GA.
Journal: J Hum Hypertens (1997): 601
Calcium transient alternans in blood-perfused ischemic hearts: observations with fluorescent indicator fura red
Authors: Wu Y, Clusin WT.
Journal: Am J Physiol (1997): H2161
IgG-induced Ca2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes
Authors: Floto RA, Mahaut-Smith MP, Somasundaram B, Allen JM.
Journal: Cell Calcium (1995): 377
Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red
Authors: Schild D, Jung A, Schultens HA.
Journal: Cell Calcium (1994): 341
Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios
Authors: Novak EJ, Rabinovitch PS.
Journal: Cytometry (1994): 135
The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells
Authors: Lew VL, Etzion Z, Bookchin RM, daCosta R, Vaananen H, Sassaroli M, Eisinger J.
Journal: Biochim Biophys Acta (1993): 152