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.

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.

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

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.

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.

Properties of SPiDER-βGal

Difference between SPiDER-βGal and comventional reagent (C12FDG)

 

Excitation and emission spectra

< Practiced conditions in application data >
Confocal microscopy Ex: 488 nm Em: 500-600nm 
Reflected Light Microscopy Ex: 545/25 nm Em: 605/70 nm
Flow Cytometer Ex: 488nm Em: 530/30 nm

Association between cellular senescence and cell cycle

Doxorubicin (DOX) acts to inhibit cell proliferation during G2/M phases of the cell cycle and induces cellular senescence. After adding DOX to A549 cells, higher histogram peaks for the G2/M phase (Cell Cycle Assay Solution Blue and Deep Red), induces cellular senescence (Cellular Senescence Detection Kit - SPiDER-βGal), and the differences in mitochondrial membrane potential (JC-1 MitoMP Detection Kit) were observed.

Markers of Senescent Cells

Senescence Markers