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Xite™ Red beta-D-galactopyranoside

Xite™ Red beta-D-galactopyranoside provides a simple and sensitive tool to detect beta-galactosidase (β-gal) activity. Compared to the existing red beta-galactosidase substrates (e.g., the commonly used resorufin beta-D-galactopyranoside), it has much better cell permeability. Xite™ Red beta-D-galactopyranoside provides a simple and sensitive tool to detect beta-galactosidase activity. Xite™ Red beta-D-galactopyranoside might be used as a simple tool for measuring cellular senescence in cells since β-gal has been identified as a reliable marker for cellular senescence. Xite™ Red beta-D-galactopyranoside enters readily cells where it gets cleaved by β-gal, producing Xite™ Red, a strongly fluorescent product. The strongly fluorescent Xite™ Red is well retained in cells, making it easy to be detected with a flow cytometer and fluorescence microscope. In addition, Xite™ Red beta-D-galactopyranoside is fixable. The red fluorescence generated by Xite™ Red beta-D-galactopyranoside can be readily combined with other color fluorescent probes such as DAPI or GFP for multicolor fluorescence analysis.

Example protocol

AT A GLANCE

Protocol summary
  1. Treat samples as desired.
  2. Prepare and add Xite™ Red beta-D-galactopyranoside working solution to samples
  3. Incubate samples at 37 °C for 15 to 45 minutes
  4. Monitor the fluorescence intensity using flow cytometer with 575/26 nm filter (PE channel) or using fluorescence microscopy with Cy3/TRITC filter set 

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Xite™ Red beta-D-galactopyranoside stock solution
Add appropriate amount of DMSO into Xite™ Red beta-D-galactopyranoside to make 2-5 mM Xite™ Red beta-D-galactopyranoside stock solution. Note: Store the unused Xite™ Red beta-D-galactopyranoside stock solution at -20 °C in single use aliquots.

PREPARATION OF WORKING SOLUTION

Xite™ Red beta-D-galactopyranoside working solution
Prepare 1-20 µM of Xite™ Red beta-D-galactopyranoside working solution in buffer of your choice. Note: Xite™ Red beta-D-galactopyranoside working solution should be used promptly. Note: The concentration of the Xite™ Red beta-D-galactopyranoside should be optimized for different cell types and conditions.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol can be used as a guideline and should be optimized according to the needs.
  1. Treat your samples as desired.
  2. Remove the treatment and wash cells with buffer of your choice such as DPBS. Note: For selectively tracking β-Gal in live cells, cells can be treated with Bafilomycin A1 for blocking endogenous β-Gal. Optimum concentration of Bafilomycin A1 may vary on type of cells.
  3. Add Xite™ Red beta-D-galactopyranoside working solution for 15-45 minutes and incubate the samples at 37 °C incubator. Note: Optimal time for incubation needs to be determined experimentally.
  4.  Remove the working solution and wash cells with buffer of your choice.
  5. Resuspend the cells in buffer of your choice and monitor the fluorescence intensity with flow cytometer using 575/26 nm filter (PE channel) or fluorescence microscope with Cy3/TRITC filter set. 

References

View all 50 references: Citation Explorer
Acridinium Benzoates for Ratiometric Fluorescence Imaging.
Authors: Wen, Min and Wang, Xijing and Wang, Ting and Sun, Yan and Fan, Mengting and Li, Min and Zhu, Junru and Zhang, Dazhi and Cui, Xiaoyan and Shan, Yongkui
Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (2020): 3247-3251
Fluorescence Signal Amplification by Using β-Galactosidase for Flow Cytometry; Advantages of an Endogenous Activity-Free Enzyme.
Authors: Nobori, Takanobu and Kawamura, Masumi and Yoshida, Ryosuke and Joichi, Taisei and Kamino, Kenta and Kishimura, Akihiro and Baba, Eishi and Mori, Takeshi and Katayama, Yoshiki
Journal: Analytical chemistry (2020): 3069-3076
Amphiphilic triphenylamine-benzothiadiazole dyes: preparation, fluorescence and aggregation behavior, and enzyme fluorescence detection.
Authors: Ishi-I, Tsutomu and Kawai, Kazuki and Shirai, Yuya and Kitahara, Ikumi and Hagiwara, Yoshinori
Journal: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society (2019): 1447-1460
Macrotheranostic Probe with Disease-Activated Near-Infrared Fluorescence, Photoacoustic, and Photothermal Signals for Imaging-Guided Therapy.
Authors: Zhen, Xu and Zhang, Jianjian and Huang, Jiaguo and Xie, Chen and Miao, Qingqing and Pu, Kanyi
Journal: Angewandte Chemie (International ed. in English) (2018): 7804-7808
Impact of plasma protein binding on cargo release by thermosensitive liposomes probed by fluorescence correlation spectroscopy.
Authors: Mittag, Judith J and Kneidl, Barbara and Preiβ, Tobias and Hossann, Martin and Winter, Gerhard and Wuttke, Stefan and Engelke, Hanna and Rädler, Joachim O
Journal: European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenst (2017): 215-223
Page updated on December 6, 2024

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Catalog Number14035
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Physical properties

Molecular weight

591.63

Solvent

DMSO

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Flow cytometer

Excitation488 nm laser
Emission575, 26 nm filter
Instrument specification(s)PE channel

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom
Expression of &beta;-gal was measured with Xite&trade; Red beta-D-galactopyranoside. 9L-LacZ cells (cells that overexpressed &beta;-gal) were incubated with Xite&trade; Red beta-D-galactopyranoside for 30 mins at 37 &deg;C. The signal was acquired with PE channel using a NovoCyte Flow Cytometer (ACEA Biosciences).
Expression of &beta;-gal was measured with Xite&trade; Red beta-D-galactopyranoside. 9L-LacZ cells (cells that overexpressed &beta;-gal) were incubated with Xite&trade; Red beta-D-galactopyranoside for 30 mins at 37 &deg;C. The signal was acquired with PE channel using a NovoCyte Flow Cytometer (ACEA Biosciences).
Expression of &beta;-gal was measured with Xite&trade; Red beta-D-galactopyranoside. 9L-LacZ cells (cells that overexpressed &beta;-gal) were incubated with Xite&trade; Red beta-D-galactopyranoside for 30 mins at 37 &deg;C. The signal was acquired with PE channel using a NovoCyte Flow Cytometer (ACEA Biosciences).