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GlycoFluor™ Series

GlycoGREEN™-βGal

[β-galactosidase activity detecting probe]

495-540 nm：Green

GlycoGREEN-βGal  is a fluorescent probe to detect β-galactosidase activity. It is a nonfluorescent substrate for β-galactosidase enzyme and fluoresces upon the reaction with the enzyme. It is a cell permeable reagent and after the reaction, generated fluorophore associates with intracellular structures. Low cytotoxicity of this reagent enables live cell imaging without interferring cellular functions.

It is suitable for lacZ reporter gene screening, for the detection of β-galactosidase activity as a cancer cell marker, and for the detection of senescence-associated β-gal (SA-β-gal) activity.

 

Available through Merck KGaA (Darmstadt, Germany ) as:
SCT025 BioTracker™ 519 Green β-Gal Dye

 

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC611 GlycoGREEN™-βGal 30 nmol × 5 SCT025 BioTracker 519 Green β-Gal Dye

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  • Product Information

    GlycoGREEN™-βGal
    Product Information

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    Principle of detection for GlycoGREEN-βGal

    GlycoGREEN-βGal is a fluorescent substrate for β-galactosidase. It is almost nonfluorescent but strongly fluoresces upon the reaction with the enzyme. Generated fluorophore associates with intracellular structures and this property is suitable for cellular imaging.  Its reaction with β-galactosidase is rapid and enables quick imaging. For example, lacZ expressing cells can be detected after incubation for 15 minutes.

     

    Properties of GlycoGREEN-βGal

    Product name
    		 target reaction Absmax (nm) FLmax (nm) ε Φ
    GlycoGREEN-βGal β-galactosidase activity irreversible 497 519 not determined 0.78

    Spectra

     

    Low cytotoxicity

    It does not show cytotoxicity in the concentrations of usual use.  Perturbation to cellular physiological functions by this reagent was not confirmed. In addition, nonspecific fluorescence signal is quite low and it strongly fluoresces upon the reaction with β-galactosidase. Therefore it is suitable to detect high-contrast fluorescence imaging in live-cell imaging.

    Live-cell imaging and cytotoxicity of GlycoGREEN-βGal （left) lacZ expressing (LacZ+) or not expressing (LacZ-) HEK293 cells were reacted with 1 μM of GlycoGREEN-βGal or other product S at 37℃ fot 15 minutes. The cells were observed in an equivalent condition. High contrast images are available. (right) Final 0, 0.1, 1, 10, 100 μM of GlycoGREEN-βGal was added to OVCAR5, an ovarian cancer cell line cells and incubated at 37℃ 5% CO2 for 24 hours. After the incubation, MTT assay was used to determine cell metabolic activities. At more tha 10 times concentations of usual use, cell activities were not damaged.

    Rapid and sensitive detection

    The reaction of GlycoGREEN-βGal is rapid and show >100 times fluorescence compared to that before the reaction. In case of lacZ overexpressing cells, 15 minutes incubation is enough to detect fluorescence. 

    Fluorescence increase of GlycoGREEN-βGal  GlycoGREEN-βGal (final 10 μM, with 0.1 % DMSO as cosolvent) in phophate buffer  (0.1 M, pH 7.4) was incubated with β-galactosidase (final 5 unit/mL) at 37℃.

  • Applications of GlycoGREEN-βGal to cell analyses

    GlycoGREEN™-βGal
    Applications of GlycoGREEN-βGal to cell analyses

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    Applications of GlycoGREEN-βGal to cell analyses

    Imaging examples of fixed cells

     

    (left) Cells were fixed after the reaction with GlycoGREEN-βGal (top) or other product S (bottom). After the reaction with 1 μM of reagents for 15 minutes, cells were fixed with 3% paraformaldehyde for 15 minutes.

    (right) Cells were fixed with 3% paraformaldehyde for 15 minutes and then, reacted with 1 μM of reagents for 15 minutes. Cells were rinsed before each observation. Cells were imaged in a same condition.

    Expression of LacZ can be detected with fluorescence of GlycoGREEN-βGal before/after the fixation. Please note that longer fixation may decrease the reactivity of β-galactosidase, and then requires longer reaction time. We recommend preliminary test in your fixation condition. We do not recommend repetitive washing of cells or cell permeabilization, those decrease teh fluorescence intensity.

     

    An example of flowcytometric analysis

    (Black) LacZ- HEK293 cells only.  (Orange)  LacZ- HEK293 cells which had reacted with 3 μM of AcidiFluor ORANGE (GC301) for 18 hours were reacted with 1 μM of GlycoGREEN-βGal for 1 hour.  (Green) LacZ+ HEK293 cells were reacted with 1 μM of GlycoGREEN-βGal  for 1 hour. Each cells were rinsed with PBS and analyzed with a flowcytometer (BD FACSVerse) one-by-one.

    Please note that generated fluorophore can leak from the positive cells and stain other negative cells. Therefore this reagent is not suitable for a flowcytometric analysis of heterogeneous cells, or for cell sorting.

FAQ

  • Q Give me a selection guide of the β-galactosidase probes.
    A

    We have three β-galactosidase probes, GlycoGREEN-βGal, GlycoGREEN-βGal, and TokyoGreen-βGal . For cell imaging, we recommend GlycoGREEN-βGal, for plate-reader assays or for enzyme kinetics measurements, we recommend TokyoGreen-βGal as a first choice.

    Sensitivity of the probe isGlycoGREEN-βGal ~  TokyoGreen-βGal > GlycoYELLOW-βGal. 
    For cell imaging, we recommend GlycoGREEN-βGal because its cell retention is relatively better, however, generated fluorophore also leaks from the cells and can be incorporated into other cells, users should carefully check the result when they observe heterogeneous cells.  

     

  • Q Can I use for the fixed samples?
    A

    You may use a similar procedure to X-gal staining for fixed cell samples, because GlycoYELLOW-βGal is also a substrate for β-galactosidase (lacZ reporter)

    We confirmed that fluorescence of GlycoYELLOW-βGal observed in live cells does not diminish by the normal process of fixation. Please refer information in the product page for details.

    Strong fixation conditions may alter the intracellular localization or may decrease the fluorescence. Please test the fixation condition in your cells and conditions.

  • Q Can I detect senescence?
    A

    Yes, using GlycoYELLOW-βGal, you can detect enhancement of β-galactosidase activity caused by senescence (SA-βGal).

    If physiological β-Gal activity in lysosomes is detected with GlycoYELLOW-βGal, then you may use bafilomycin A1 for alkalization of lysosomes and inhibit physiological β-Gal activity. You may not need to use alkalization reagent if you do not detect physiological β-Gal.

  • Q My question is not in this FAQ list.....
    A

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

D. H. Ho, D. Nam, M. K. Seo, S. W. Park, W. Seol, I. Son (2021)
Oxid. Med. Cell. Longev. 2021:9969842. DOI: 10.1155/2021/9969842

D. H. Ho, W. Seol, I. Son (2019)
Cell Cycle 18:467-475  DOI: 10.1080/15384101.2019.1577666

D. Asanuma, M. Sakabe, M. Kamiya, K. Yamamoto, J. Hiratake, M. Ogawa, N. Kosaka, P. L. Choyke, T. Nagano, H. Kobayashi, Y. Urano (2015)
Nat. Commun. 6: 6463 DOI:10.1038/ncomms7463

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