Products / Goryo Chemical, Inc.

HOME > Products & Services > MetalloFluor™ Series > [Specific detection of copper(I) ion in living cells] CopperGREEN

[Specific detection of copper(I) ion in living cells] CopperGREEN

  • High specificity to copper ion: Low reactivity with other trace metal ions and reactive oxygen species (ROS).
  • High S/N: Fluorescence increase of >100 times can be observed upon reaction with copper (I) ion.

Features

Copper is one of the trace metals essential for living organisms. Copper is an essential cofactor for enzyme activities of cytochrome c, superoxide dismutase, and tyrosinase. CopperGREEN is a fluorescent probe to detect CuI, which is a dominant redox state of copper in an intracellular reducing environment. It is suitable for live cell imaging.

Principle of the measurement

CopperGREEN is a colorless material with no fluorescence. When CopperGREEN chelates CuI ion, it is easily hydrolyzed to generate fluorescent material with excitation maximum, ~480 nm and emission maximum, 510 nm. Fluorescence remains after mild fixation because its reaction is irreversible.

Principle of the measurement

Reactivity of CopperGREEN

Reactivity of CopperGREEN

Figure 1. Reactivity of CopperGREEN to trace metals and reactive oxygen species (ROS).

Fluorescence of CopperGREEN increases only in the presence of CuI ion.

 

Measurement conditions
  • Fluorescence of 1 µM CopperGREEN in 0.05 M HEPES buffer (pH7.2) were measured after incubation of with the following reagents at 37℃, 120 min. Either metal ions of 20 μM supplemented with 2 mM glutathione, or ROS generating reagents as described later was used. In CuII condition, glutathione was not used.
  • Fluorescent intensities were measured at 510 nm, with excitation at 470 nm by using a microplate reader (TECAN Infinite M200 Pro), with excitation slit width of 9 nm, emission slit width of 20 nm.
ROS generating conditions
  • H2O2: 300 µM H2O2
  • OCl: 300 µM NaOCl
  • OH: 20 µM Fe(ClO4)2, 200 µM H2O2
  • none: 0.05 M HEPES buffer (pH 7.2) as a control.

Fluorescence spectra and reactivity of CopperGREEN

Fluorescence spectra and reactivity of CopperGREEN

Figure2. Spectrum and reaction properties of CopperGREEN  (left) Spectrum change of CopperGREEN after reaction with CuI (Emmax = 510 nm). (right) Reaction properties indicate that CopperGREEN reacts with CuI ions in molar ratio of 1:1. Reaction takes ~90 minutes to saturate.

 

Measurement conditions
  • (left) Fluorescence spectra of 5 µM CopperGREEN before and after the reaction with CuI. measured with HITACHI F-2700 fluorescence spectrophotometer with the slit width of 2.5 nm and the photomultiplier voltage of 700V. CopperGREEN was reacted with 100 μM [CuI(CH3CN)4]PF6 at 37℃ for 2 hours in a buffer solution of 50 mM HEPES (pH 7.2), 2 mM glutathione, and 0.5% DMSO as a cosolvent.
  • (right, top) Fluorescence intensity of 1 µM CopperGREEN depending on the concentration of CuI, which was measured using a microplate reader (TECAN Infinite M200 Pro) with excitation wavelength of 470 nm, emission wavelength of 510 nm, excitation bandwidth of 9 nm, emission bandwidth of 20 nm. CopperGREEN was reacted with [CuI(CH3CN)4]PF6 at 37℃ for 2 hours in a buffer solution of 50 mM HEPES (pH 7.2), 2 mM glutathione, and 0.1% DMSO as a cosolvent.
  • (right, bottom) Time course of 1 µM CopperGREEN reaction with 100 μM CuI. Fluorescence was measured by using HITACHI F-2700 fluorescence spectrophotometer with excitation wavelength of 470 nm, emission wavelength of 510 nm, slit width of 5 nm, photomultiplier voltage of 700V.

An example live-cell imaging of copper

An example live-cell imaging of copper

Figure 3. Imaging of copper ions incorporated in HeLa cells.  Fluorescence image of CopperGREEN (green) was overlaid to DIC image.

  • HeLa cells cultured in a culture medium (DMEM + 8% FBS, penicillin/streptomycin) supplemented with 200 µM CuCl2 were washed with PBS containing 200 µl EDTA, 2 times to remove extracellular copper ions.
  • Cells were stained in a medium containing 5 μM CopperGREEN for 3 hours, at 37℃ 5% CO2.
  • For fluorescence observation, the medium was exchanged with HBSS to reduce the background.

※ To reduce nonspecific staining, add 10 mM NH4Cl or 100 nM bafilomycin A1 to the medium 30 minutes before the staining and during the staining. This treatment inhibit acidification of lysosomes and endosomes which reduces nonspecific oxidization of CopperGREEN in acidic environments. (10 mM NH4Cl was used in the above example.)

※ Microscope: Leica DMI 6000 CS, objective lens: 40×

Reference
Masayasu Taki, Shohei Iyoshi, Akio Ojida, Itaru Hamachi, Yukio Yamamoto
J. Am. Chem. Soc., 2010, 132, 5938–5939 doi: 10.1021/ja100714p
Price
Code No. Product Size Price Protocol MSDS
GC 902 CopperGREEN 50 nmol × 5 $498 
  • Donwload
    458KB
  • Donwload
    256KB

Contact Information

For further information or questions on our products, please contact us, E-mail: info_itnl@goryochemical.com