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AcidiFluor™ Series

AcidiFluor™ ORANGE

[For live cell imaging of lysosomes]

570-590 nm：Orange

AcidiFluor ORANGE is a lysosome-staining fluorescence probe, made of fluorophore which fluorescence greatly increases in acidic environment fused with lysosome-localizing chemical structure. Users can easily image acidic lysosomes of living cells just by adding this reagent to the extracellular fluid.  Its bright and photostable fluorescence enables timelapse imagings for a longer time spans.

 

Available through Merck KGaA (Darmstadt, Germany ) as:
SCT 019 BioTracker™ 560 Orange Lysosome Dye

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC301 AcidiFluor™ ORANGE 10 μg × 20
GC3011 AcidiFluor™ ORANGE 10 μg × 10 SCT019 BioTracker 560 Orange Lysosome Dye

Downloads

  • Protocol

  • Flyer

  • SDS

  • Product Information

    AcidiFluor™ ORANGE
    Product Information

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    Properties of AcidiFluor ORANGE

    Product name target reaction pKa
    		 Absmax (nm) FLmax (nm) ε* Φ**
    AcidiFluor ORANGE pH reversible 5.1 535 560 N.D. 0.73

    * molar extinction coeffient, not determined.
    ** Maximal quantum yield. It changes with pH.

    Spectra

    Fluorescence spectra of AcidiFluor Orange measured in phosphate buffer of pH 5.0 or that of pH 7.4, respectively. pH 5.0 buffer corresponds to the condition in the acidic organelles, and pH 7.4 corresponds to the physiological cytoplasmic conditions. Fluorescent intensity of AcidiFluor Orange at pH 5.0 increased 50 fold to that in the pH 7.4 buffer. ( λex 532 nm / λem 568 nm )

     

    Goryo Chemical, Inc. commercialized AcidiFluor™ ORANGE under the guidance of Prof. Kenzo Hirose (Graduate School of Medicine, The University of Tokyo).  AcidiFluor ORANGE is licensed from Tokyo University. The development of this product was supported by Japan Science and Technology Agency program “Development of Systems and Technology for Advanced Measurement and Analysis”.

  • Imaging examples using AcidiFluor ORANGE

    AcidiFluor™ ORANGE
    Imaging examples using AcidiFluor ORANGE

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    A multicolor imaging example using AcidiFluor ORANGE

    HeLa cells expressing Mitochondria-GFP were stained with AcidiFluor ORANGE and Hoechst 33342. Lysosomes images stained with AcidiFluor ORANGE (Orange), nuclei stained with Hoechst 33342 (blue), and Mitochondria stained with GFP (green) were overlayed.

     

    Comparison with other products by a photocleaching test

    Samples were continuously illuminated for 180 seconds and imaged with confocal microscopy.  Significant pohotobleach of LysoTracker® Green DND-26 and LysoTracker® Red DND-99 were observed, whereas fluorescence of AcidiFluor ORANGE remained. LysoSensor™ Green DND-189 leaked to cytoplasm after the continuous excitation light irradiation, suggesting it is not suitable for continuous observation. 

  • Imaging example of degranulation with AcidiFluor ORANGE

    AcidiFluor™ ORANGE
    Imaging example of degranulation with AcidiFluor ORANGE

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    Imaging of deglanulation by using AcidiFluor ORANGE

    Visualizing deglanulation of RBL-2H3 cells

    Granules were stained with AcidiFluor ORANGE (1 µM in DMEM) and extracellular fluid phase was visualized by a highly soluble fluorophore, Alexa Fluor® 647, carboxylic acid. Degranulation was induced by the addition of ionomycin and observed by confocal microscopy. Fluorescence intensity of AcidiFluor ORANGE decreased upon degranulation, while the fluorescence intensity of Alexa Fluor 647 increased. The graph shows fluorescence intensity change of the glanule.
    AcidiFluor ORANGE can be thus used for degranulation imaging.

    Movie

     

     

    Other movie

    Degranulation Imaging of RBL-2H3 Cell (rat basophilic leukemia cell) stained with AcidiFluor ORANGE. Fluorescence images are overlayed with differential interference contrast images. Degranulation was induced by the addition of ionomycin and observed by confocal microscopy.

    Procedure

    1. 10 µg of AcidiFluor ORANGE in a vial was dissolved with 13.4 µL of dimethylsulfoxide (DMSO) to prepare 1 mM solution.
    2. Diluted the 1 mM solution with culture medium to prepare1 μM staining medium.
    3. Removed the culture medium from cell culture dish and washed twice with medium.
    4. Added the staining medium to the dish and cultured for 2 hours at a 37 ℃, 5% CO2 condition.
    5. After the staining, rinced once with medium, twice by HBSS buffer. Replaced to HBSS buffer and observed by fluorescence microscopy.

FAQ

  • Q Tell me the fluorescence intensity at pH less than 3.
    A

    Intensity of AcidiFluor ORANGE is almost constant at the pH 3 or less.

  • Q Can I use for fixed samples?
    A

    No, basically, it cannot be applied to fixed cells. Acidic pH of lysosomes and endosomes are kept by the activity of living cells and fixed (dead) cells do not keep the acidic pH.

    On the other hand it could be possible to detect localization of the probes by soaking fixed cells into an acidic buffer, because localization of AcidiFluor ORANGE, which has a structure to be localized in lysosomes, antibody-labeled AcidiFuor ORANGE-NHS and HaloTag labeled HaloTag AcidiFluor ORANGE ligand can be kept for a while after fixation.

     

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

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

T. Karasawa, A. Kawashima, F. Usui-Kawanishi, S. Watanabe, H. Kimura, R. Kamata, K. Shirasuna, Y. Koyama, A. Sato-Tomita, T. Matsuzaka, H. Tomoda, S. Y. Park, N. Shibayama, H. Shimano, T. Kasahara, M. Takahashi (2018)
Arterioscler. Thromb. Vasc. Biol. 38: 744–756 DOI:10.1161/ATVBAHA.117.310581

S. Kitazawa, S. Nishizawa, H. Nakagawa, M. Funata, K. Nishimura, T. Soga, T. Hara (2017)
Cancer Sci. 108: 1185-1193 DOI:10.1111/cas.13240

K. Kitakaze, Y. Mizutani, E. Sugiyama, C. Tasaki, D. Tsuji, N. Maita, T. Hirokawa, D. Asanuma, M. Kamiya, K. Sato, M. Setou, Y. Urano, T. Togawa, A. Otaka, H. Sakuraba, K. Itoh (2016)
J. Clin. Invest. 126: 1691-1703 DOI:10.1172/JCI85300

A. Hayashi, D. Asanuma, M. Kamiya, Y. Urano, S. Okabe (2016)
Neuropharmacology 100: 66-75 DOI:10.1016/j.neuropharm.2015.07.026

D. Asanuma, Y. Takaoka, S. Namiki, K. Takikawa, M. Kamiya, T. Nagano, Y. Urano, K. Hirose (2014)
Angew. Chem. Int. Ed. Engl. 53: 6085-6089 DOI:10.1002/anie.201402030

M. Isa, D. Asanuma, S. Namiki, K. Kumagai, H. Kojima, T. Okabe, T. Nagano, K. Hirose (2014)
ACS. Chem. Biol. 9: 2237-2241 DOI:10.1021/cb500654q

R. Watanabe, N. Soga, D. Fujita, K. V. Tabata, L. Yamauchi, S. H. Kim, D. Asanuma, M. Kamiya, Y. Urano, H. Suga, H. Noji (2014)
Nat. Commun. 5: 4519 DOI:10.1038/ncomms5519

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