Cellular Senescence Assay

∼ Feature ∼

  1. Quantify SA-βgal
  2. Applicable for Living Cell and Tissues
  3. Staining time 30 min.



 


Kit content (1 plate) : SPiDER-βGal x 1, Bafilomycin A1 x 1

Storage Condition : 0-5oC

Shipping Condition : ambient temperature


Product Description

DNA damages of the normal cells are caused by repeated cell division and oxidative stress. Cellular Senescence, a state of irreversible growth arrest, can be triggered in order to prevent DNA-damaged cells from growing. Senescence-associated β-galactosidase (SA-β-gal), which is overexpressed in senescent cells, has been widely used as a marker of cellular senescence. Although X-gal is a well known reagent to detect SA-β-gal, these are following disadvantages: 1) requirement of fixed cells due to the poor cell-permeability, 2) low quantitative capability because of the difficulty of the determination of visual difference between stained cells and not stained cells, 3) requirement of a long time of staining.
Cellular Senescence Detection Kit - SPiDER-βGal allows to detect SA-β-gal with high sensitivity and ease of use. SPiDER-βGal is a new reagent to detect β-galactosidase which possesses a high cell-permeability and a high retentivity inside cells. SA-β-gal are detected specifically not only in living cells but also fixed cells by using a reagent (Bafilomycin A1) to inhibit endogenous β-galactosidase activity. Therefore, SPiDER-βGal can be applied to quantitative analysis by flow cytometry.


      

          Fig.1 Detection mechanism of senescent cells by SPiDER-βGal       Fig.2 Excitation and emission spectra of SPiDER-βGal after reaction with β-galactosidase



Co-staining of SA- β-gal and DNA Damage marker in WI-38 cells




Fig.3 Cell cultures subcultured one time (passage 1) and ten times (passage 10) were stained. a) SA- β-gal produced by senescent cells are labelled, b) γ-H2AX marker indicates presence of DNA damage, c) DAPI indicates the location of the nuclear, and d) merged image of three markers





Procedure:

  1. Passage 1 and 10 of WI-38 were used. The procedure was followed as the manual within the kit.
  2. Add 4% PFA/PBS to the cells and incubate for 15 minutes at room temperature
  3. Wash the cells 3 times with PBS
  4. Add 0.1% Triton X-100/PBS to cells and incubate for 30 minutes at room temperature
  5. Wash the cells 3 times with PBS
  6. Add 1% BSA/PBS to the cells and incubate for 1 hour at the room temperature
  7. Add anti- γ-H2AX antibody (rabbit) diluted with 1% BSA/PBS to the cells and incubate at 4℃ overnight
  8. Wash the cells 3 times with PBS
  9. Add Anti- rabbit secondary antibody (Alexa Fluor 647) diluted with 1% BSA/PBS to the cells and incubate at room temperature for 2 hours
  10. Wash cells 3 times with PBS
  11. Add 2 μg/ml DAPI (code: D523) diluted with PBS to the cells and incubate for 10 minutes at room temperature
  12. Wash cells 3 times with PBS and observe under a confocal microscope



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

Fluorescence imaging of SA-β-gal

1. WI-38 cells (5×104 cells/dish, MEM, 10% fetal bovine serum, 1% penicillin-streptmycin) of passage number 0 and 12 were seeded respectively in a µ-dish 35 mm (ibidi) and cultured overnight in a 5% CO2 incubator.

2. The cells were washed with 2 ml of HBSS once.

3. Bafilomycin A1 working solution (1 ml) was added to the culture dish, and the cells were incubated for 1 hour in a 5% CO2 incubator.

4. SPiDER-βGal working solution (1 ml) was added to the culture dish, and the cells were incubated for 30 minutes in a 5% CO2 incubator.

5. After the supernatant was removed, the cells were washed with 2 ml of HBSS twice.

6. HBSS (2 ml) were added and the cells were observed by confocal fluorescence microscopy (Excitation: 488 nm Emission (wavelength/band pass): 550/50 nm).



Fig.4 Fluorescence imaging of SA-β-Gal in WI-38 cells

A. Passage 0, B. Passage 12

(green: SPiDER-βGal, blue: Hoehst 33342)





Quantitative analysis of SA-β-gal positive cells by flow cytometry

1. WI-38 cells (1×105 cells/dish, MEM, 10% fetal bovine serum, 1% penicillin-streptmycin) of passage number 1 and 12 were seeded respectively in a µ-dish 35 mm (ibidi) and cultured overnight in a 5%CO2 incubator.

2. The cells were washed with 2 ml of HBSS once.

3. Bafilomycin A1 working solution (1 ml) was added to the culture dish, and the cells were incubated for 1 hour in a 5%CO2 incubator.

4. SPiDER-βGal working solution (1 ml) was added to the culture dish, and the cells were incubated at for 30 minutes in a 5%CO2 incubator.

5. After the supernatant was removed, the cells were washed with 2 ml of HBSS twice.

6. The cells were harvested by trypsin and resuspended in MEM (10% fetal bovine serum, 1% penicillin-streptmycin).

7. The cells were observed by a flow cytometer (Excitation: 488 nm, Emission: 515-545 nm).





Fig.5 Quantification of SA-β-Gal positive WI-38 cells






Detection of SA-β-gal in the Tissue Sample

Reference paper using Dojindo’s SPiDER-β-gal to detect SA-β-gal (code: SG02) in 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.



Code# Unit Size Ships in* Price Quantity
SG04-01
1 plate
1-2 business days $200.00
SG04-03
3 plate
1-2 business days $450.00

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