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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. Applicable for Living Cell and Fixed Tissue
  3. Staining time 30 min.

For Tissue sample, please use SPiDER-ßGal (Click Here for the product page)


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

Product Description
Simple Procedure
Applicable for Living and Fixed Cells
Detection with Flow Cytometry
Markers of Senescent Cells
Co-staining with DNA Damage marker: Live cells
Co-staining with DNA Damage marker: Fixed cells
Confocal Quantitative Image Cytometer
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


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


Preparation of SPiDER-βGal working solution

Dilute the SPiDER-βGal DMSO stock solution 2,000 times *1 with McIlvaine buffer (pH 6.0).
*1 Fixation and permeablization could leads to lower sensitivity (Figure 1), if you need higher signals,dilute the SPiDER-βGal DMSO stock solution 500 – 1,000 times with the McIlvaine buffer (Figure 2).

Preparation of McIlvaine buffer (pH 6.0)

Mix 0.1 mol/l citric acid solution (3.7 ml) and 0.2 mol/l sodium phosphate solution (6.3 ml). Confirm the pH is 6.0. If the pH is not 6.0, adjust the pH by adding either citric acid solution or sodium phosphate solution. Dilute this buffer 5 times with ultrapure water.

Staining procedure (35 mm dish)

1. Prepare cells on 35 mm dish for assay and culture the dish at 37℃ overnight in a 5% CO2 incubator.
2. Remove the culture medium. Add 2 ml of 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 3 minutes *2.
*2 Avoid a longer treatment period, which leads to decrease in SA-β-gal activity.
3. Remove the supernatant, and wash the cells 3 times with 2 ml of PBS.
4. Add 2 ml of SPiDER-βGal working solution and incubate at 37℃ for 30 minutes*3.
*3 We recommend not to use a 5% CO2 incubator for fixed cell experiments. If incubation is done in a 5% CO2 incubator, the pH of the buffer may become acidic. Acidic pH results in higher background from the endogenous β-galactosidase activity and it would be difficult to distinguish between normal cells and senescent cells.
5. After removing the supernatant, wash the cells twice with PBS.
6. Add 0.1% Triton X-100/PBS to cells and incubate for 30 minutes at room temperature.
7. Wash the cells twice with PBS.
8. Add 1% BSA/PBS to the cells and incubate for 1 hour at the room temperature
9. Add anti- γ-H2AX antibody (mouse) diluted with 1% BSA/PBS to the cells and incubate at 4℃ overnight.
10. Wash the cells 3 times with PBS.
11. Add anti- mouse secondary antibody (Cy5) diluted with 1% BSA/PBS to the cells and incubate at room temperature for 1 hour.
12. Wash cells twice with PBS and observe under a fluorescence microscope.


Quantification with confocal quantitative image cytometer

In the conventional method of X-gal, SA-β-gal-positive cells are counted under microscope and calculate the percent of the senescent cells by compared with total cells. The SA-β-gal-positive cells were stained with this kit and analyzed using confocal quantitative image cytometer CQ1(Yokogawa Electric Corporation).



The difference of SA-β-gal-positive cells ratio were shown in WI-38 cells depending on the number of passage. The data was quickly analysed with the confocal quantitative image cytometer compared with the manually counting procedure with X-gal staining method.


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.
3) A. Park, I. Tsunoda and O. "Heat shock protein 27 promotes cell cycle progression by down-regulating E2F transcription factor 4 and retinoblastoma family protein p130", J. Biol. Chem.., 2018, doi: 10.1074/jbc.RA118.003310 .
4)R. Tanino, Y. Tsubata, N. Harashima, M. Harada and T. Isobe,"Novel drug-resistance mechanisms of pemetrexed-treated non-small cell lung cancer", Oncotarget., 2018, 9, (24), 16807.
5)Y. Kitahiro, A. Koike, A. Sonoki, M. Muto, K. Ozaki and M. Shibano. ,"Anti-inflammatory activities of Ophiopogonis Radix on hydrogen peroxide-induced cellular senescence of normal human dermal fibroblasts.", J Nat Med ., 2018, 72, 905.

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.