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.

Recent work from Dr. Kim et al. at Mayo Clinic used our Cellular Senescence Detection Kit – SPiDER-βGal to evaluate cellular senescence in endothelial cells. They did staining of SA-βGal in cells in atherosclerotic renal artery stenosis (ARAS) and co-stained the cells with CD31 which is a marker of endothelilal cells. They showed that ARAS + Elamipretide* treatment slightly improved endothelial cell senescence. Unlike commercial available probes used for detection of β-Galactosidase, SPiDER-βGal contained in the kit possesses high intracellular retention. The key feature of this product is that it can be used to co-stain SA-β-Gal and other markers. Our kit is a useful tool for cellular senescence research.*Elamipretide: mitochondria-targeted peptide

For more information on data, please refer to the publication below:S. R. Kim, A. Eirin, X. Zhang, A. Lerman and L. O. Lerman, “Mitochondrial Protection Partly Mitigates Kidney Cellular Senescence in Swine Atherosclerotic Renal Artery Stenosis.”, Cell. Physiol. Biochem. ., 2019, 52, 617.

Simple Procedure

After Bafilomycin A1 Working Solution is added, SPiDER-βGal Working Solution is added without removing Bafilomycin A1 Working Solution.

Difference between X-Gal method and Cellular Senescence Detection Kit – SPiDER-βGal IOur kit is applicable to both living and fixed cells. However, X-Gal method is only applicable to dead cells as shown below:

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-βGalOur 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 temperature3. Wash the cells 3 times with PBS4. Add 0.1% Triton X-100/PBS to cells and incubate for 30 minutes at room temperature5. Wash the cells 3 times with PBS6. Add 1% BSA/PBS to the cells and incubate for 1 hour at the room temperature7. Add anti- γ-H2AX antibody (rabbit) diluted with 1% BSA/PBS to the cells and incubate at 4℃ overnight8. Wash the cells 3 times with PBS9. Add Anti- rabbit secondary antibody (Alexa Fluor 647) diluted with 1% BSA/PBS to the cells and incubate at room temperature for 2 hours10. Wash cells 3 times with PBS11. Add 2 μg/ml DAPI (code: D523) diluted with PBS to the cells and incubate for 10 minutes at room temperature12. 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 solutionDilute 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 temperature9. 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 cytometerIn 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

Comparison with other product

Doxorubicin-treatment A549 cells stained with each reagent were incubated for 30 min or 120 min and the resulting fluorescence images were compared. SPiDER-ßGal (Item# SG04) had higher fluorescent intensity than other product.

・Epifluorescence Microscope (Fixed A549 cells)

・Confocal Microscopy (Fixed A549 cells)

Fluorescence imaging of SA-β-gal1. 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) and 1 mg/ml Hoechst 33342 (1 µl) were mixed. Then the mixture solution 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 cellsA. Passage 0, B. Passage 12(green: SPiDER-βGal, blue: Hoehst 33342)

Quantitative analysis of SA-β-gal positive cells by flow cytometry1. 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

How to Prepare a Positive Control using Drug TreatmentSenescence induction (Doxorubicin-treatment WI-38 cells)1. Seed WI-38 cells (1×10⁶ cells/dish, MEM, 10% fetal bovine serum, 1% penicillin-streptmycin) of passage number 3 in a 10 cm culture dish and culture at 37 ℃ overnight in a 5% CO₂ incubator.2. Remove the culture medium, and wash the cells with 10 ml of PBS once.3. Prepare for 0.2 μmol/L of Doxorubicin with serum-free MEM. In case, if serum free media is not available, serum contained media may be used.4. Add Doxorubicin (10 mL) to the dish and culture at 37 ℃ for 3 days in a 5% CO₂ incubator.5. Remove the supernatant, and wash the cells with 10 ml of PBS once.6. Add MEM (10% fetal bovine serum, 1% penicillin-streptmycin) to the dish and culture at 37 ℃ for 3 days in a 5% CO₂ incubator.7. Remove the culture medium, and wash the cells with 10 ml of PBS once.8. Trypsinize the cells with and without Doxorubicin treatment.

Fixed cell imaging1. Prepare cells in a 8-well ibidi for assay and culture the cells at 37℃ overnight in a 5% CO₂ incubator.2. Remove the culture medium. Wash the cells with PBS once. Add 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 3 minutes.3. Remove the supernatant. Wash the cells with PBS twice.4. Mix SPiDER-βGal working solution (2 mL) and 1mg/mL Hoehst 33342 (2 μl). Add the mixture solution (200 μl) into a well, and incubate at 37℃ for 30 minutes.

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. Remove the supernatant. Wash the cells with PBS twice.6. Observe the cells under a fluorescence microscope.

ReferenceSenescence induction in serum-free media1. Leontieva, O.V.; Blagosklonny.M.V. “DNA damaging agents and p53 do not cause senescence in quiescent cells, while consecutive re-activation of mTOR is associated with conversion to senescence.” Aging (Albany NY). 2010, 2, 924-935.Senescence induction in serum-contained media 2. Demaria, M.; O’Leary, M.N.; Chang, J., et al. “Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.” Cancer Discov. 2017, 7, 165-176.

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.2016033282. 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. Yoshie, “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, 905.6. R. Uchida, Y. Saito, K. Nogami, Y. Kajiyama, Y. Suzuki, Y. Kawase, T. Nakaoka, T. Muramatsu, M. Kimura and H. Saito , “Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging”, NPJ Aging Mech Dis., 2018,doi：10.1038/s41514-018-0031-5.7. S. R. Kim, A. Eirin, X. Zhang, A. Lerman and L. O. Lerman, “Mitochondrial Protection Partly Mitigates Kidney Cellular Senescence in Swine Atherosclerotic Renal Artery Stenosis.”, Cell. Physiol. Biochem. ., 2019, 52, 617.8. Webber L, Yujra V, Vargas P, et al. “Interference with the bromodomain epigenome readers drives p21 expression and tumor senescence.”, Cancer Letters. 2019; 461: 10-20.9. H. Ise, K. Matsunaga, M. Shinohara and Y. Sakai, “Improved Isolation of Mesenchymal Stem Cells Based on Interactions between N-Acetylglucosamine-Bearing Polymers and Cell-Surface Vimentin “, Stem Cells Int ., 2019, 4341286, 13.10. Wang X, Qu M, Li J, et al. “Induction of Fibroblast Senescence During Mouse Corneal Wound Healing.”, Invest Ophthalmol Vis Sci. 2019; 60: 3669-3679.11. Y. Nakatani, H. Kiyonari and T. Kondo, “Ecrg4 deficiency extends the replicative capacity of neural stem cells in a Foxg1-dependent manner.”, Development., 2019, 146, (4), 18.12. Y. S. Ryu, K. A. Kang, M. J. Piao, M. J. Ahn, J. M. Yi, G. Bossis, Y. M. Hyun, C. O. Park and J. W. Hyun , “Particulate matter-induced senescence of skin keratinocytes involves oxidative stress-dependent epigenetic modifications”, Exp. Mol. Med. ., 2019, 51, 108.13. E. M. Angela Ibler, E. Mohamed, L. N. Kathryn, A. B. Natalia, F. E. K. Sherif and H. Daniel, “Typhoid toxin exhausts the RPA response to DNA replication stress driving senescence and Salmonella infection”, Nat Commun., 2019, 10, 4040