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SPiDER-ßGal

Item # Unit Size
SG02-10
20 µg × 3

For Research Use Only Products

β-galactosidase Assay

∼ Feature ∼

  1. A Newly Fluorogenic β-galactosidase Detection Probe
  2. Applicable for Living Cell and Tissues
  3. Detect β-galactosidase at Single-Cell Resolution
  4. Quantify SA-βgal in Cellular Senescence Assay



Storage Condition : 0-5oC
Shipping Condition : ambient temperature
CAS# : 1824699-57-1

Product Description

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 SPiDER-βGal. SPiDER-βGal ideally possesses cell-permeability and the ability to retain in intracellular region.1)

By the enzymatic reaction, SPiDER-βGal 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, SPiDER-βGal allows a single-cell analysis because it does self-immobilizing to the intracellular proteins.


Usage Examples:

Fluorescence microscopic detection of β-galactosidase-expressed cells


  1. HEK cells at 5 × 105 cells/ml (500 μl) and HEK/LacZ cells at 5 × 105 cells/ml (500 μl) were seeded in a 35 mm dish in DMEM (10% fetal bovine serum, 1% penicillin-streptmycin) and cultured overnight in a 5% CO2 incubator at 37oC.
  2. The cells were washed with 2 ml of Hanks’ HEPES buffer twice.
  3. SPiDER-βGal working solution (2 ml) was added to the culture dish. The cells were then incubated for 15 minutes at 37oC.
  4. After the supernatant was removed, the cells were washed Hanks’ HEPES buffer (2 ml) twice.
  5. Hanks’ HEPES buffer (2 ml) were added and the cells observed under a fluorescence microscope. (Fig. 3A)
  6. After the supernatant was removed, 4% paraformaldehyde (PFA) /PBS solution (2 ml) was added to the culture dish. The cells were then incubated for 15 minutes at room temperature.
  7. After 4% PFA/PBS solution was removed, the cells were washed Hanks’ HEPES buffer (2 ml) twice.
  8. Hanks’ HEPES buffer (2 ml) were added and the cells observed under a fluorescence microscope. (Fig. 3B)


Flow cytometric detection of β-galactosidase-expressed cells

  1. HEK cells at 5 × 105 cells/ml (500 μl) and HEK/LacZ cells at 5 × 105 cells/ml (500 μl) were mixed in a microtube.
  2. SPiDER-βGal DMSO stock solution (1 μl) was added to the tube. The cells were then incubated 15 minutes at 37oC .
  3. The cells were analyzed under a flow cytometer. (488 nm excitation, 530/30 nm bandpass filter)

β-galactosidase-expressed cells (HEK/LacZ cells) were clearly differentiate from HEK cells in flow cytometry data analysis.



Reference

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, doi: 10.1002/anie.201603328
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, doi: 10.1002/1873-3468.12271
3) Y. Nakamura, A. Mochida, T. Nagaya, S. Okuyama, F. Ogata, P. L. Choyke, H. Kobayashi, "A topically-sprayable, activatable fluorescent and retaining probe,SPiDER-βGal for detecting cancer; Advantages of anchoring to cellular proteins after activation", Oncotarget, 2017, doi: 10.18632/oncotarget.17080

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.







Imaging of a Fixed Tissue of a Drosophila
Formalin fixed drosophila generative cells were stained by SPiDER-βGal and DAPI. The β-galactosidase, which is expressed at the nucleus of drosophila generative cells, could be obtained by SPiDER-βGal.



Data was kindly provided by Dr. T. Nakamura, at University of Kumamoto, Institute of Molecular Embryology and Genetics.







Imaging of a Pancreas of a Mouse
SPiDER-βGal and Salmon-Gal were evaluated in the detectability of β-galactosidase on a pancreas of a mouse. SPiDER-βGal was able to detect the difference of the expression point of β-galactosidase more clearly than Salmon-Gal.



Data was kindly provided by Dr. S. Kume, at Tokyo Institute of Technology, School of Bioscience and Biotechnology.







Cell Senescence Assay with SPiDER-βGal by fluorescent microscopy and flow cytometry
SA β-Gal in WI-38 cells induced by hydrogen peroxide was detected with SPiDER-βGal by fluorescent microscopy. SA β-Gal expression correlates with expression of other cellular senescence marker γ-H2AX. The increased expression of SA β-Gal was measured by flow cytometry.

        



Mouse Tissue

1) Live Staining

・Dissect lacZ-expressed tissue from mouse , and cut it to the appropriate size.

・Incubate in OPTI-MEM containing 10-20 μmol/l SPiDER-βGal at 37 degree in 5% CO2. (It is possible to fix with 4% paraformaldehyde (PFA) after staining.)

2) Fixed Tissue Staining

・Dissect lacZ-expressed tissue from mouse , and cut it to the appropriate size.

・Fix with a solution containing 1% PFA and 0.2% GA , and then incubate in PBS containing 10-20 μmol/l SPiDER-βGal for 60 minutes at room temperature.


Reference paper using Dojindo’s SPiDER-β-gal for the tissue sample of diabetic mouse model was published.

<Condition Tissue Samples were Labelled>

Tissue sample was sliced into thin pieces after rapid freezing. The sliced samples were incubated in 4% Paraformaldehyde at room temperature for 20 minutes. First the samples were washed in PBS. Then, 20 μmol/l SPiDER-βGal was added and was incubated for 1 hour at 37℃. The samples were washed in PBS and observed under microscope.

For more detail, please refer to the publication:

T. Sugizaki, S. Zhu, G. Guo, A. Matsumoto, J. Zhao, M. Endo, H. Horiguchi, J. Morinaga, Z. Tian, T. Kadomatsu, K. Miyata, H. Itoh & Y. Oike, "Treatment of diabetic mice with the SGLT2 inhibitor TA-1887 antagonizes diabetic cachexia and decreases mortality", Nature Partner Journal:Aging and Mechanisms of Disease., doi:10.1038/s41514-017-0012-0.


Drosophila Tissue

1) Live Staining

・Dissect lacZ-expressed tissue from Drosophila larvae , and then incubate in medium containing 10-20 μmol/l SPiDER-βGal for 20-30 minutes at room temperature.

・If you would like to staining nuclear, please add 16 μmol/l Hoechst 33342 together. It is possible to fix with 4% paraformaldehyde (PFA) after staining.

2) Fixed Tissue Staining

・Dissect lacZ-expressed tissue from Drosophila larvae , and then fix for 20 minutes in 4% PFA in PBS.

・After washing in PBS, incubate in PBS containing 10-20 μmol/l SPiDER-βGal for 20-30 minutes at room temperature.

3) Combination with Immunostaining

・Dissect lacZ-expressed tissue from Drosophila larvae , and then fix for 20 minutes in 4% PFA in PBS.

・Wash with PBS-T for 3 times, 5 minutes each.

・Dissolve primary-antibody in PBS-T and incubate for 30 minutes.

・Wash with PBS-T for 3 times, 5 minutes each.

・Incubate in PBS containg 10 μmol/l SPiDER-βGal, 16 μmol/l Hoechst 33342 and secondary antibody for 30 minutes.

・Wash with PBS for 3 times, 5 minutes each.


Q1:Is there comparison data with X-Gal? What is an advantage over X-Gal?

A1 : SPiDER-βGal allows stain of a sample with a shorter time and without fixing the sample.

Please check the comparison data below. There is comparison data between SPiDER-βGal and X-Gal for tissue staining.

【Mouse Kidney】

Refer to Figure S14 and 15 on the Supporting Information.

【Mouse Salivary Gland】

Refer to Figure S14 and 15 on the Supporting Information.

【Mouse Brain】

Refer to Figure S16 and 17 on the Supporting Information.


Q2:What is an advantage over commercially available β-Galactosidase detection probe?

A2:SPiDER-βGal has higher cell permeability and intracellular retention than any other commercially available probe, which allows stain of a sample with a shorter time and lower concentration for mammalian cells. Please check the Selection Guide for more details.


Q3:Can I use SPiDER-βGal for tissue staining?

A3:Yes, SPiDER-βGal can be used for tissue staining. We have experimental data with the following samples:

【Drosophila Tissue】

Refer to Figure S9, 12 and 13 on the Supporting Information.

【Mouse Kidney】

Refer to Figure S14 and 15 on the Supporting Information.

【Mouse Salivary Gland】

Refer to Figure S14 and 15 on the Supporting Information.

【Mouse Brain】

Refer to Figure S16 on the Supporting Information.


Q4:Can I fix a sample after staining with SPiDER-βGal?

A4:Yes, the sample stained with SPiDER-βGal can be fixed with 4% PFA or methanol.


Q5:Can I stain a fixed sample with SPiDER-βGal?

A5:Yes, SPiDER-βGal can be used for the fixed sample. We have experimental data for HEK cell, drosophila and mouse tissue. However, since fixation causes lower β-galactosidase activity, please optimize the condition for fixation.


Q6:How many time can I assay with 1 kit (20 μg×3) ?

A6:If the final concentration is prepared at 1 μmol/l for cell staining, you can do approximately fifty 35mm dishes, fifty 8-chamber plates and ten 96- well plates.


Q7:Is there information regarding cytotoxicity?

A7:Cytotoxicity of SPiDER-βGal to HEK-lacZ(+) cells and HEK cells was measured with Cell Counting Kit-8.

【Cytotoxicity assay with HEK-lacZ(+) cells and HEK cells】

Refer to Figure S8 on the Supporting Information.


Q8:How long is the Working solution stable?

A8:You can’t store the working solution. Please prepare the working solution prior to use.


Q9:What buffer can I use for preparing the Working solution?

A9:You can use PBS, Hanks' HEPES and HBSS etc.


Q10:What is the recommended filter?

A10: Fluorescence microscopy: Ex.550/25 nm, Em.605/70 nm

Flow Cytometry: Ex.488 nm, Em.530/30 nm