94433 β-Galactosidase Stain

Product number 94433

SPiDER-βGal

The gene of β-galactosidase from E. coli is widely used as a reporter gene assay marker. Although X-gal is well known reagent to detect β-galactosidase in cell or tissue samples, the assay using these reagents require to fix cells or tissues due to the poor cell-permeability. In addition, so far developed the assay using fluorescence reagents cannot clearly differentiate β-galactosidase-expressed cells or regions.

To overcome these issues, Urano, Kamiya and co-workers have successfully developed a stain ideally possesses cell-permeability and the ability to retain in intracellular region.1)

By the enzymatic reaction, the β-Galactosidase stain immediately forms a quinone methide that acts as electrophile when proteins containing nucleophilic functional groups nearby the molecules. By the probe undergoes the reaction with a protein, the conjugates become fluorescent compounds. Thus, it allows a single-cell analysis because it does self-immobilizing to the intracellular proteins.
Excitation Maximum: 530 nm (± 10 nm)
Emission Maximum: 550 nm (± 10 nm)


Feature of SPiDER-βGal

Easy Procedure: not require fixation of cells or tissue
High Sensitivity: single-cell resolution



Figure 1. Staining principle

Application Data

Reporter gene assay



Figure 2. Live imaging of drosophila tissue

Living drosophila tissue was incubated with 10 µmol/l SPiDER-βGal and 16 µmol/l Hoechst 33342 for 20-30 min, observed with confocal microscope. Only β-galactosidase expressed cells was detected with SPiDER-βGal.

Data was kindly provided by Dr. Y. Urano, at University of Tokyo, Graduate School of Medicine.
 

Cellular senescence



Figure 3. Detection of senescence-associated β-galactosidase (SA-β-Gal)

WI-38 cells were subcultured 1 or 11 times and stained with SPiDER-βGal. The strong fluorescence through SA-β-Gal was observed in the passage 11 cells.
 

Comparison with other substrates


Figure 4.
Comparison of x-gal and SPiDER-βGal

 

General protocol:

Preparation of 1 mmol stain DMSO stock solution:
Add 35 μl of DMSO to a tube of stain solution (20 μg) and dissolve it with pipetting.
*Store the stock solution at -20°C.

Preparation of 1 μmol/l stain working solution:
Dilute the stain DMSO stock solution with Hanks′ HEPES buffer to prepare 1 μmol/l stain working solution.
*Hanks′ HEPES buffer is recommended to maintain cell condition.

Staining:

  1. Prepare cells for the assay
  2. Discard the culture medium and wash the cells with Hanks′ HEPES buffer twice
  3. Add an appropriate volume of stain working solution
  4. Incubate at 37oC for 15 minutes
  5. Observe the cells under a fluorescence microscope or by a flow cytometer

*After staining, the cells can be observed even without washing. However, you can wash it as needed.

References:
1) T. Doura, M. Kamiya, F. Obata, Y. Yamaguchi, T. Y. Hiyama, T. Matsuda, A. Fukamizu, M. Noda, M. Miura, Y. Urano, "Detection of LacZ-Positive Cells in Living Tissue with Single-Cell Resolution.", Angew Chem Int Ed Engl., 2016, 55(33), 9620.
2) H. Omori, S. Ogaki, D. Sakano, M. Sato, K. Umeda, N. Takeda, N. Nakagata, S. Kume, "Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development.", FEBS Lett., 2016, 590(16), 2584.