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Cal Red™ R525/650 potassium salt

Fluorescence emission spectra of Cal Red™ R525/650 (calcium bound).
Fluorescence emission spectra of Cal Red™ R525/650 (calcium bound).
Fluorescence emission spectra of Cal Red™ R525/650 (calcium bound).
Graph illustrates signal-to-noise ratio (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.
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Physical properties
Dissociation constant (Kd, nM)330
Molecular weight~1000
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
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Molecular weight
Dissociation constant (Kd, nM)
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 well excited at 488 nm with two emissions at 525 nm and 650 nm. Upon binding calcium, the emission signal of Cal Red™ R525/650 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.

Example protocol


Sample protocol to determine Kd values

Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in
fluorescence microplate readers) of the indicators in solutions with precisely known free Ca2+ concentrations. Calibration
solutions can be used based on 30 mM MOPS EGTA Ca2+ buffer. In general, water contains trace amount of calcium ion. It
is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM
calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca2+

A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA

B. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl2

To determine either the free calcium concentration of a solution or the Kd of a single-wavelength calcium indicator,
the following equation is used: 

[Ca]free = Kd[F ─ Fmin]/[Fmax ─ F]

Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, Fmin is the
fluorescence intensity in the absence of calcium and Fmax is the fluorescence intensity of the calciumsaturated probe. 

The dissociation constant (Kd) is a measure of the affinity of the probe for calcium. The calcium-binding and
spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration
solutions. In situ response calibrations of intracellular indicators typically yield Kd values significantly higher than in vitro
determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca2+ buffers in the presence of
ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin
or Triton® X-100 can be used to expose the indicator to the controlled Ca2+ levels of the extracellular medium. 



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
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
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
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
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
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
Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers
Authors: Kurebayashi N, Harkins AB, Baylor SM.
Journal: Biophys J (1993): 1934