Simple Procedure

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

Why is Bafilomycin A1 added?

Endogenous β-galactosidase existing in living cells interfere with selective detection of SA-β-Gal. Bafilomycin A1 is an inhibitor of ATPase in lysosome. pH in lysosome is kept neutral by adding Bafilomycin A1. Cellular Senescence Detection Kit - SPiDER-βGal contains Bafilomycin A1 which allows to detect SA-β-Gal selectively. Bafilomycin A1 is utilized for living cell assays only. Bafilomycin A1 is not used in fixed cells because intracellular pH is controlled with the buffer.

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

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

For plate reader detection, please refer Cellular Senescence Plate Assay Kit - SPiDER-βGal.

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.

Markers of Senescent Cells

SA-β-gal detection for tissue

In this published article, SA-β-gal was detected using SPiDER-βGal on tissue samples from a diabetic model mouse.

Note: SPiDER-βGal [Code: SG02] is used in this staining.

After slicing the frozen tissue, it was fixed with 4% paraformaldehyde for 20 minutes at room temperature. Then it was washed with PBS and observed.

For details of the experimental operation and data, refer to Reference 2) below.

SA-β-gal detection for T cells (floating cells)

The research group by professor Masakatsu Yamashita at Ehime University Graduate School of Medicine has shown that a protein called Menin controls T cell exhaustion, aging, and maintains normal immune function.

By using this kit, they confirmed that SG03 has the ability to detect induced cell senescence by stimulating TCR (T cell receptor) in the presence of interleukin 2 (IL-II) in naive CD8+ T cells that are knocked out Menin.

Staining Conditions

① SPiDER-βGal method

② X-gal method

*Data was kindly provided by Masakatsu Yamashita, at Ehime University Graduate School of Medicine

Experiment Example: Cellular Senescence with Metabolic shift

NAD(+) levels decline during the aging process, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies*. Here, we try to redemonstrate this phenomenon in the doxorubicin (DOX)-induced cellular senescence model with a comprehensive analysis of our products.

*S. Imai, et al., Trends Cell Biol, 2014, 24, 464-471

Related Products

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.