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Cellular Senescence Detection Kit - SPiDER-ßGal

Item # Unit Size
SG03-10
10 Assays

For Research Use Only Products

Cellular Senescence Assay

∼ Feature ∼

  1. Quantify SA-βgal
  2. pplicable for Living Cell and Fixed Tissue
  3. Staining time 30 min.


Kit content : SPiDER-βGal x 1, Bafilomycin A1 x 1
Storage Condition : 0-5oC
Shipping Condition : ambient temperature

  • Product Description
  • Simple Procedure
  • Difference between X-Gal method and Cellular Senescence Detection Kit - SPiDER-βGal I: Applicable for Living Cell and Fixed Tissue
  • Difference between X-Gal method and Cellular Senescence Detection Kit - SPiDER-βGal II: Easier Detection
  • Markers of Senescent Cells
  • Co-staining of SA- β-gal and DNA Damage marker in WI-38 cells
  • Recommended Filter

  • 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.


    Simple Procedure



    Difference between X-Gal method and Cellular Senescence Detection Kit - SPiDER-βGal I

    Our kit is applicable to both living and fixed cells. However, X-Gal method is only applicable to dead cells as shown below:



    Difference between X-Gal method and Cellular Senescence Detection Kit - SPiDER-βGal II

    Our kit allows quantification of SA-β-Gal using flow cytometry.



    Markers of Senescent Cells


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


    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


    Recommended Filter


    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, doi: 10.1002/anie.201603328
    2) 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.

    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.


    Are there any advices when observing the senescent cells?
    Lipofuscin is a fluorescent pigment that accumulates in a variety of cell types with age. Lipofuscin consists of autofluorescent granules and may results in high background for fluorescence microscopy. In order to achieve accurate SA-β-gal activity assay in senescent cells, we recommend to prepare samples without SPiDER-βGal staining. Please compare fluorescence intensity of both cells with or without SPiDER-βGal staining.

    > For Flow Cytometry Detection
    Step 1. Prepare senescent cells and non-senescent cells. Measure MFI (Mean Fluorescence Intensity) of samples below.
    [Senescent cells]
    Sample A: The cells stained with SPiDER-βGal
    Sample B: The cells without SPiDER-βGal staining
    [Non-senescent cells]
    Sample A’: The cells stained with SPiDER-βGal
    Sample B’: The cells without SPiDER-βGal staining

    Step 2. Calculate SA-β-gal activity (senescent cells) with the following formula
    SA-β-gal activity (senescent cells) = MFI of Sample A - MFI of Sample B

    Step 3. Calculate SA-β-gal activity (non-senescent cells) with the following formula
    SA-β-gal activity (non-senescent cells) = MFI of Sample A’ - MFI of Sample B’
    • Determine the SA-β-gal activity by comparing the SA-β-gal activity between senescent cells and non-senescent cells.
    • Change of SA-β-gal activity associated with senescence = (Value from Step 2- value from Step 3)

    >For Microscopy
    Step 1. Prepare senescent cells without SPiDER-βGal staining and observe fluorescent image.
    Step 2. Adjust detection sensitivity in microscopy to reduce background autofluorescence of lipofuscin.
    Step 3. Observe fluorescent image of senescent cells and non-senescent cells under the settled condition in step 2.