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[A fluorescent probe to quantify intracellular glutathione]QuicGSH3.0

Based on intramolecular FRET mechanism, intracellular glutathione (GSH) concentrations can be quantified from the ratio of fluorescent intensities between two wavelengths.

  • The dissociation constant (Kd = 3.0 mM) is optimum for measuring intracellular GSH concentrations.
  • Quick and reversible response to the GSH concentration changes.
  • Relatively slow photobleaching and low cytotoxicity enable stable measurements of intracellular GSH concentrations.

Principle of measurement and probe characteristics

Usually, it is not difficult to quantify an analyte in a test tube using a fluorescent probe for detection. In contrast, it is difficult to measure the intracellular concentration of the analyte, because there is usually no simple method to determine the concentration of the analyte in the environment of the cell independent of probe concentration.

Based on a intramolecular FRET mechanism (FRET = fluorescence resonance energy transfer), QuicGSH3.0 has been applied for measuring the intracellular GSH concentrations regardless of the probe concentrations. QuicGSH3.0 is a molecule formed with an orange fluorescent dye (T) and a deep-red fluorescent dye (S). At a low GSH concentration, green light excitation of T, leads to an energy transfer to S and a subsequent emission of deep-red fluorescent light. At a high GSH concentration, the energy transfer hardly occurs, so the probe emits the orange fluorescent light from the emission of T.

Principle of measurement and probe characteristics

The mechanism enables determination of intracellular GSH concentration from a ratio of fluorescence intensities between the two wavelengths, regardless of the probe concentrations. QuicGSH3.0 has an optimum dissociation constant to GSH (Kd = 3.0 mM) for quantifying intracellular GSH concentrations which has been considered to be in a millimolar range. Because the ratio rapidly changes upon the GSH concentration change, QuicGSH3.0 can be used for real-time monitoring of intracellular GSH concentration changes.

Absorption and fluorescence spectra

Absorption and fluorescence spectra
Absorbance spectra (left) and emission spectra (right) of QuicGSH3.0

The emission maximum (λem) is 625 nm in the absence of GSH, whereas it shifts to 582 nm upon addition of GSH. Upon excitation with a green light at 520-550 nm, the ratio of fluorescence intensities between 582 nm (Fluorescent wavelength 1) and 625 nm (Fluorescent wavelength 2) changes as the GSH concentration changes.

Fluorescence intensity ratio against GSH concentration
Fluorescence intensity ratio against GSH concentration

2 µM QuicGSH3.0 (in 0.2 M phosphate buffer, pH 7,4 containing 5% DMSO) was excited at 550 nm after adding GSH of different concentrations. Fluorescent intensities were measured at 580 nm and 616 nm. Finally, the fluorescent intensity ratio (R = F580/F616) was plotted against GSH concentrations.

Rapid and reversible reaction

QuicGSH3.0 rapidly reacts with GSH to shift its λem from 625 to 580 nm. λem also returns quickly to 625 nm when GSH is quenched.

QuicGSH3.0 reactivity

QuicGSH3.0 reactivity
2 µM QuicGSH3.0 (in 0.2 M phosphate buffer, pH 7.4, containing 5% DMSO as cosolvent) was incubated with GSH at 27ºC. At 30 minutes later, a thiol scavenger, N-ethylmaleimide (NEM) was added at 25 mM. Fluorescence intensity was recorded every 60 seconds with a microplate reader (TECAN infinite M200Pro). The ratio of fluorescence intensity at 580 nm and 620 nm were calculated. (λex: 550 nm, λem: 580 nm and 620 nm, n=3 ± S.D.)

An example of live-cell imaging

An example of live-cell imaging

2 µM QuicGSH3.0 (containing 0.01% Pluronic F-127) was added to A549 cells and incubated for 10 minutes to be incorporated. The intracellular concentration of GSH (right) was calculated from images captured in the two wavelength ranges. 

Refer the protocol document, detailed protocols for customers, and references for the details.

References
Keitaro Umezawa, Masafumi Yoshida, Mako Kamiya, Tatsuya Yamasoba, Yasuteru Urano (2017)
Nat. Chem. 9: 279-286. DOI: 10.1038/nchem.2648
Price
Code Product Size Price Protocol MSDS
A401-1 QuicGSH3.0 25 nmol × 5 $698.00 Download
343KB
Download
441KB
A401-2 25 nmol × 2 $340.00
※ 5 vials kit (A401-1) is recommended for users who try this reagent first, especially for microscope users, because relatively larger amount of reagent is required for the calibration.

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