Calcium Green

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Calcium Green


Calcium GreenTM (CaG) denotes a family of extrinsic fluorophores applied for measurement of Ca2+ concentration with mitochondrial preparations. This dye fluoresces when bound to Ca2+. When measuring mitochondrial calcium uptake it is possible to observe the increase of the CaG signal upon calcium titration, followed by the decrease of CaG signal due to the uptake.

Abbreviation: CaG

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Calcium Green in high-resolution respirometry (HRR)


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.
Set the Gain for Amp sensor to 1000 and light intensity (Amp polarization voltage [mV]) to 500. These values can be modified by the user if needed (e.g., if a different CaG concentration is used).

The fluorescent dye

Calcium GreenTM is a registered trademark and available from Thermo Fisher Scientific (formerly: Invitrogen) in several formulations. For measuring mitochondrial calcium uptake a membrane impermeant formulation must be chosen (e.g. C3737).
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.

Preparation of CaG solution

Calcium Green from Thermo Fischer Scientific (former Invitrogen): C3737 (Calcium Green™-5N, Hexapotassium Salt, cell impermeant); 0.5 mg vial, store at -20°C.
Preparation of 1 mM stock solution (dissolved in H2O):
  1. Dissolve the complete vial of CaG (0.5 mg) in 419 µL of deionized H2O.
  2. Divide into 40 µL portions into 0.2 mL Eppendorf tubes (protect from light, use dark tubes preferably).
  3. Store frozen at -20 °C protected from light.
Titration into 2 mL O2k-Chamber: 4 µL of 1 mM CaG stock solution, final concentration of 2 µM CaG. This concentration can be adjusted if needed.

Additional information

  • EGTA should be titrated in the media, in the presence of CaG, to chelate possible contaminants, before Ca2+ titration. For isolated mitochondria, we recommend 10-15 µM (to be added before the sample). For permeabilized cells, our preliminary tests indicated ~70 µM as the optimal EGTA concentration (which can be added after the addition of the cells and before digitonin permeabilization).
  • We tested stepwise CaCl2 titrations of 5 µM, every 6 min or when the Ca2+ was taken up. The CaCl2 concentration will depend on the sample concentration.
  • We recommend measure calcium uptake capacity in LEAK state. However, a small concentration of ADP may be needed to stimulate calcium uptake, depending on the tissue.
  • 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.
  • A KCl-based buffer can be used, but it will not stimulate respiration as much as MiR05.
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.

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 Smart-Module is operated through the amperometric Fluo-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.

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.


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


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
Cecatto 2022 Abstract Bioblast20225.1. «10+5»
Cecatto Cristiane
Cecatto Cristiane, Cardoso LHD, Gnaiger E (2022) Mitochondrial calcium uptake capacity is lower than calcium retention capacity in the presence and absence of cyclosporin A. Bioblast 2022: BEC Inaugural Conference. In: »Watch the presentation«
Nickel 2018 IOC1302018Defects in mitochondrial calcium uptake precede defects of the respiratory chain in X-linked Barth syndrome cardiomyopathy.MouseHeartCardiovascular

MitoPedia methods: Fluorometry