Price and Availability

Kit content (1 plate) : 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

. 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.


Price List

Item # Description/Size Availability Qty Break Price Quantity
SG04-01
1 plate
1-2 business days 1 $200.00
SG04-03
3 plate
1-2 business days 1 $450.00
SG04-10
10 plate
1-2 business days 1 CALL

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