Calcium Green

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Calcium Green


Calcium Green denotes a family of extrinsic fluorophores applied for measurement of Ca2+ concentration.

Abbreviation: CaG

MitoPedia methods: Fluorometry 

Sources and applications

Calcium GreenTM is a registered trademark and available from Life Technologies (former Invitrogen) in several formulations.
All fluorophores of the Calcium Green family have the same fluorophoric moiety in their molecular structure and therefore near identical optical properties. They differ in their KD values and are suitable for measuring different ranges of calcium concentrations.
  • Calcium Green 1: KD = 190 nM
  • Calcium Green-2: KD = 550 nM
  • Calcium Green-5N: KD = 14000 nM
In Chapter 19 of The Molecular Probes Handbook, available here you can see how to choose the right Fluorescent Ca2+ Indicators and also information about chelators, calibration buffers, ionophores and Cell-Loading Reagents. If you intend to use O2k-Fluo Smart-Module you need to choose Calcium green as a probe. Calcium Green hexapotassium salt is a simpler choice since it does not require loading and washing the dye in the sample.
See Calcium for method-independent considerations related to measuring calcium levels, covering e.g.
  • Considerations before starting a project involving calcium measurements;
  • Ca2+ buffering;
  • Calculation of free Ca2+ concentrations;
  • Ca2+ calibration buffers.

MiR05 cannot be used as a buffer for calcium measurements since it contains a high concentration of EGTA and other components that can chelate calcium signal For more information see Naszai 2019 Sci Rep.

Colour of media and chemicals used
For all fluorometric techniques, special care must be taken to avoid colored chemicals that might affect the fluorescence signal (excitation and emission wavelength spectra).
With CaG fluorophore, some uncouplers that present a yellow color might affect the signal.

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O2k-Open Support

MitoPedia O2k and high-resolution respirometry: O2k-Open Support 

Ca measurement

In high-resolution respirometry the CaG is used with the O2k-FluoRespirometer with O2k-Fluo Smart-Module or O2k-Fluo LED2-Module selecting the Fluorescence-Sensor Blue/Filter Set MgG / CaG. Please see these pages for specific aspects of fluorescence measurements using the O2k.
Use black stoppers with black cover-slips to exclude disturbances by external light sources.
Before the experiments, switch off the illumination of the chambers (in [Oroboros O2k] \ [ O2k control ], or [F7]).
The three Calcium Green fluorophores and Magnesium Green share the same fluorophore moiety in their molecular structure, therefore the optical properties are very similar and the same fluorescence sensor and filter set can be used. Since the used concentration of Calcium Green might vary according to the application, the optimum light intensity and amplification used may be different from those for Magnesium Green. However, the values recommended for Magnesium Green will provide a good starting point.
On the fluorophores producers homepage detailed product information is available
General information on measuring calcium, e.g.

Signal and output

  1. Signal: The O2k-Fluo LED2-Module is operated through the amperometric (Amp)-Channel of the O2k, with electric current (ampere [A]) as the primary signal.
  2. Output: The focus of the output with Calcium Green is on Type A: Concentration, and Type C: Force, potential.


  • Use of Calcium Green-5N with the other instruments in parallel with oxygen consumption measurements with the O2k.


-See: Defective fluorescence module

Publications: CaG O2k-FluoRespirometer

Deline 2021 Biochim Biophys Acta Bioenerg2021Deline ML, Grashei M, van Heijster FHA, Schilling F, Straub J, Fromme T (2021) Adenylate kinase derived ATP shapes respiration and calcium storage of isolated mitochondria. Biochim Biophys Acta Bioenerg 1862:148409.MouseLiver
Duplaquet 2020 Elife2020Duplaquet L, Leroy C, Vinchent A, Paget S, Lefebvre J, Vanden Abeele F, Lancel S, Giffard F, Paumelle R, Bidaux G, Heliot L, Poulain L, Furlan A, Tulasne D (2020) Control of cell death/survival balance by the MET dependence receptor. Elife 9:e50041.HumanHEKCell death
Spinazzi 2019 Proc Natl Acad Sci U S A2019Spinazzi M, Radaelli E, Horré K, Arranz AM, Gounko NV, Agostinis P, Maia TM, Impens F, Morais VA, Lopez-Lluch G, Serneels L, Navas P, De Strooper B (2019) PARL deficiency in mouse causes Complex III defects, coenzyme Q depletion, and Leigh-like syndrome. Proc Natl Acad Sci U S A 116:277-86.MouseNervous systemNeurodegenerative
Naszai 2019 Sci Rep2019Nászai A, Terhes E, Kaszaki J, Boros M, Juhász L (2019) Ca(2+)N it be measured? Detection of extramitochondrial calcium movement with high-resolution FluoRespirometry. Sci Rep 9:19229.RatLiver
Liepinsh 2016 Biochem J2016Liepinsh E, Makrecka-Kuka M, Volska K, Kuka J, Makarova E, Antone U, Sevostjanovs E, Vilskersts R, Strods A, Tars K, Dambrova M (2016) Long-chain acylcarnitines determine ischaemia/reperfusion-induced damage in heart mitochondria. Biochem J 473:1191-202.RatHeartIschemia-reperfusion
Elustondo 2014 Biochim Biophys Acta2014Elustondo PA, Negoda A, Kane CL, Kane DA, Pavlov EV (2014) Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore. Biochim Biophys Acta 1847:231–40.RatLiverPermeability transition
Abstracts: CaG O2k-FluoRespirometer
Nickel 2018 IOC1302018Defects in mitochondrial calcium uptake precede defects of the respiratory chain in X-linked Barth syndrome cardiomyopathy.MouseHeartCardiovascular