Cellular Senescence Detection Kit - SPiDER BlueSG07 Product Manual

DNA damage in normal cells may be caused by repeated cell division and oxidative stress. Cellular senescence, a state of irreversible growth arrest, can be triggered in response to DNA-damage. Senescence-associated β-galactosidase (SA-β-gal), which is overexpressed in senescent cells, has been widely used as a marker of cellular senescence ^{1, 2)}.
This kit allows for the detection of SA-β-gal with high sensitivity and ease of use. Because SPiDER Blue emits blue fluorescence after reacting with SA-β-gal in fixed cells, it is possible to co-stain with green or red fluorescent probes and fluorescent labeled antibodies for immunostaining.


Fig. 1 Detection mechanism of senescent cells by SPiDER Blue

Fig. 2 Excitation and emission spectra of SPiDER Blue after reaction with β-galactosidase 

1 plate (6-well plate)

• SPiDER Blue              10 µl × 1
• Assay Buffer              13 ml × 1

Store at -20°C.

• Micropipettes
• Phosphate buffered saline (PBS)
• Paraformaldehyde (PFA) / PBS solution (4%)

Preparation of SPiDER Blue working solution

Dilute 20 mmol/l SPiDER Blue with Assay Buffer to prepare a 15 μmol/l SPiDER Blue working solution.

• In order to prepare 2 ml of the SPiDER Blue working solution, dilute 1.5 μl of SPiDER Blue with 2 ml of Assay Buffer.
• The optimal concentration of SPiDER Blue depends on the cells; consider a range of 10-20 μmol/l as a guide.
• Please use the SPiDER Blue working solution on the same day as preparation.

 

1. Seed cells in a dish and culture them at 37°C in an incubator equilibrated with 95% air and 5% CO₂.
2. Remove the culture medium and wash the cells with PBS.
3. Add 4% paraformaldehyde (PFA) / PBS solution to the cells and incubate at room temperature for 30 minutes.
4. Discard the 4% PFA / PBS solution and wash the cells with PBS.
5. Add 15 µmol/l SPiDER Blue working solution and incubate at 37°C for 30 minutes.
   • We do not recommend using 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 a higher background from the endogenous β-galactosidase activity and could make it difficult to distinguish between normal cells and senescent cells.
6. Remove the working solution, and wash the cells with PBS.
7. Add PBS and observe the cells under a fluorescence microscope.

Fluorescence imaging of SA-β-gal in doxorubicin-treated A549 cells

1. A549 cells (1 × 10⁶ cells/dish, DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on a 10-cm dish and cultured at 37°C overnight in a 5% CO₂ incubator.
2. The medium was removed and the cells were washed with serum-free DMEM once. Doxorubicin (DOX) solution (0.2 μmol/l in serum-free DMEM) was added, and the cells were cultured at 37°C for 3 days in a 5% CO₂ incubator.
3. The DOX solution was removed, and the cells were washed with serum-free DMEM once. DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin was added, and the cells were cultured at 37°C for 3 days in a 5% CO₂ incubator.
4. The trypsinized DOX-treated and untreated A549 cell suspension (1 × 10⁵ cells/ml, DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded (200 μl/well) on a μ-slide 8-well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
5. The medium was removed, and the cells were washed with PBS once. A 4% paraformaldehyde (PFA) / PBS solution was added to the cells and incubated at room temperature for 30 minutes.
6. The 4% PFA / PBS solution was removed, and the cells were washed with PBS once.
7. SPiDER Blue working solution (15 µmol/l in Assay Buffer) was added to the cells and incubated at 37°C for 30 minutes.
8. The working solution was removed, and the cells were washed with PBS once. PBS was added, and the cells were observed by confocal fluorescence microscopy (40×).

Fig. 3 Fluorescence imaging of SA-β-gal

CTRL: Normal condition, DOX.: Senescent condition
Filter sets: 405 nm (Ex), 400–500 nm (Em)

Fluorescence imaging of SA-β-gal in non-senescent and senescent WI-38 cells with different passage numbers.

1. WI-38 cells (1 × 10⁶ cells/dish, MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) of passage number 4 and 12 were seeded in 10-cm dishes and cultured at 37°C overnight in a 5% CO₂ incubator.
2. The trypsinized WI-38 cell suspension (1 × 10⁵ cells/ml, MEM containing 10% fetal bovine serum and 1% penicillin/streptomycin) were seeded (200 μl/well) on a μ-slide 8-well plate (ibidi) and cultured at 37°C overnight in a 5% CO₂ incubator.
3. The medium was removed, and the cells were washed with PBS once. A 4% paraformaldehyde (PFA) / PBS solution was added to the cells and incubated at room temperature for 30 minutes.
4. The 4% PFA / PBS solution was removed, and the cells were washed with PBS once.
5. SPiDER Blue working solution (15 µmol/l in Assay Buffer) was added to the cells and incubated at 37°C for 30 minutes.
6. ​The working solution was removed, and the cells were washed with PBS once. PBS was added, and the cells were observed by confocal fluorescence microscopy (40×).

Fig. 4 Fluorescence imaging of SA-β-gal

P=4: Passage number 4, P=12: Passage number 12
Filter sets: 405 nm (Ex), 400–500 nm (Em)

1. Dimri, G. P. et al., Cell Biology, 1995, 92, 9363–9367.
2. Park, A. M. et al., J. Biol. Chem., 2018, 293(41), 15815-15826.