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Cal-670™, potassium salt

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. Cal-670™ is a near infrared (NIR) calcium indicator with maximum emission at ~675 nm. It can be well excited with the red lasers at 633 nm or 647 nm with a moderate calcium affinity of Kd ~853 nM. Cal-670™ is one of the very few calcium indicators that can be potentially used for in vivo imaging since it has a NIR fluorescence.

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

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+ concentrations.
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. The fluorescence can be measured at Ex/Em = 650/675 nm. (Cutoff = 665 nm)

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of Cal-670™, potassium salt to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM62.973 µL314.863 µL629.727 µL3.149 mL6.297 mL
5 mM12.595 µL62.973 µL125.945 µL629.727 µL1.259 mL
10 mM6.297 µL31.486 µL62.973 µL314.863 µL629.727 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Citations

View all 12 citations: Citation Explorer
All-optical crosstalk-free manipulation and readout of Chronos-expressing neurons
Authors: Soor, Navjeevan S and Quicke, Peter and Howe, Carmel L and Pang, Kuin T and Neil, Mark AA and Schultz, Simon R and Foust, Amanda J
Journal: Journal of physics D: Applied physics (2019): 104002
All-Optical Crosstalk-Free Manipulation and Readout of Chronos-expressing Neurons
Authors: Soor, Navjeevan Singhh and Quicke, Peter and Howe, Carmel L and Pang, Kuin Tian and Neil, Mark and Schultz, Simon and Foust, Am and a Joy, undefined
Journal: Journal of Physics D: Applied Physics (2018)
Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)
Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945
Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781

References

View all 53 references: Citation Explorer
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
Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
Journal: Gen Physiol Biophys (2004): 357
Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1
Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and microphysiometry
Authors: Patel H, Porter RH, Palmer AM, Croucher MJ.
Journal: Br J Pharmacol (2003): 671
A sensitive method for the detection of foot and mouth disease virus by in situ hybridisation using biotin-labelled oligodeoxynucleotides and tyramide signal amplification
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187
Page updated on December 6, 2024

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Physical properties

Dissociation constant (Kd, nM)853

Molecular weight

1587.99

Solvent

Water

Spectral properties

Excitation (nm)

667

Emission (nm)

680

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Cal-670 was incubated with buffer that contains different concentration of free Ca2+. The fluorescence was monitored on fluorimeter GeminiXS (Molecular Device) at 650 nm/ 675 nm.
Cal-670 was incubated with buffer that contains different concentration of free Ca2+. The fluorescence was monitored on fluorimeter GeminiXS (Molecular Device) at 650 nm/ 675 nm.
Cal-670 was incubated with buffer that contains different concentration of free Ca2+. The fluorescence was monitored on fluorimeter GeminiXS (Molecular Device) at 650 nm/ 675 nm.