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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

For cell sample by plate reader, Click Here for the product page.
For tissue sample, Click Here for the product page.
For cell sample by fluorescence microscopy or flow cytometry, see information down below.

∼ Feature ∼

  1. Quantify SA-βgal
  2. Applicable for Living Cell and Fixed Tissue
  3. Staining time 30 min.


Technical Manual Supplemental Manual


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.


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 I

Our 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-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.


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.

<Staining Conditions>

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 SG04 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



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.



Quantification with confocal quantitative image cytometer

Co-staining and analysis with other senescent markers

Live cell staining of SA-β-gal with SPiDER-βGal after cell fixation and membrane permeabilization, and quantification with a confocal quantitative imaging cytometer using WI-38 cells immunofluorescently stained for DNA damage marker γ-H2AX.

Culture plate: Chamber Objective Lens:10 times Emission Detector 405 nm (Hoechst 33342): Cyan 488 nm (SPiDER-βGal): Green 640 nm (Alexa Fluor 647-anti-γ-H2AX): Magenta Field of view: 6 fields Analysis area color: SA-β-gal positive WI-38 cells


Quantitative analysis – scatter plot Quantitative analysis was performed using SPiDER-βGal as a scatter plot of the fluorescence value of senescent cells (X axis) using SA-β-gal as an index, and the ratio of the γ-H2AX area to the nucleus area of ​​each cell (Y axis).

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

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# SG03) had higher fluorescent intensity than other product.


・Epifluorescence Microscope (Fixed A549 cells)



・Confocal Microscopy (Fixed A549 cells)



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) 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 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

How to Prepare a Positive Control using Drug Treatment

Senescence 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 imaging

1. 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.

      Reference

      Senescence induction in serum-free media

      1) 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.

      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.

      No. Sample Instrument Reference
      1) Cell
      (HEK)
      Gene
      (LacZ)
      Fluorescence microscope,
      FCM
      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, 55, 33
      2) Tissue
      (mouse adipose tissues)
      Fluorescence microscope 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., 2017, doi:10.1038/s41514-017-0012-0.
      3) Cell
      (HSP27-knockdown)
      Protein
      (Ki67, cyclin B1)
      Fluorescent microscope 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) Cell
      (A549)
      Fluorescence microscope,
      FCM
      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) Cell
      (NHDF)
      Fluorescence microscope 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) Tissue
      (intestinal epithelial organoids from aged mice)
      Fluorescent microscope 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) Tissue
      (frozen section of kidney)
      Fluorescent microscope 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) Cell
      (HN6, HN12, HN13)
      FCM Liana P. Webber, Veronica Q. Yujra, Pablo A. Vargas, Manoela D. Martins. Cristiane H. Squarize, Rogerio M. Castilho, "Interference with the bromodomain epigenome readers drives p21 expression and tumor senescence", Cancer Letters., 2019, doi.org/10.1016/j.canlet.2019.06.019.
      9) Cell
      (UE7T-13)
      FCM 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) Tissue
      (mouse corneal stroma)
      FCM X. Wang, M. Qu, J. Li, P. Danielson, L. Yang and Q. Zhou, "Induction of Fibroblast Senescence During Mouse Corneal Wound Healing.", Invest. Ophthalmol. Vis. Sci., 2019, 60, (10), 3669.
      11) Cell
      (VZ/SVZ)
      FCM 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) Cell
      (HaCaT, HEK001)
      Fluorescent microscope 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) Cell
      (HT1080)
      Fluorescent microscope 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.
      14) - (Review) FCM B.L. Torres, A. Estepa-Fernandez, M. Rovira, M. Oraez, M. Serrano, R. Martinez-Manez and F. Sancenon,"The chemistry of senescence.", The chemistry of senescence., 2019, (3), 426-411
      15) Cell
      (HepG2)
      Fluorescent microscope 三谷塁一, "肝臓のミトコンドリア活性化に及ぼす大豆イソフラボンの効果とその分子機構に関する研究", 大豆たん白質研究, 2019, 21
      16) Cell
      (PC12)
      Fluorescent microscope N. Wang, H. Wang, L. Li, Y. Li and R. Zhang, "β-Asarone Inhibits Amyloid-β by Promoting Autophagy in a Cell Model of Alzheimer's Disease.", Front Pharmacol., 2020, 10, 1529
      17) Cell
      (A2780)
      FCM Z. Wang, J. Gao, Y. Ohno, H. Liu and C. Xu, "Rosiglitazone ameliorates senescence and promotes apoptosis in ovarian cancer induced by olaparib.", Cancer Chemother Pharmacol., 2020.
      18) Tissue
      (frozen section of mouse kidney)
      Fluorescent microscope,
      FCM
      J. H. Cho, E. Kim, Y. Son, D. Lee, Y. S. Park, J. H. Choi, K. Cho, K. Kwon and J. Kim, "CD9 Induces Cellular Senescence and Aggravates Atherosclerotic Plaque Formation.", Cell Death Differ., 2020, doi: 10.1038/s41418-020-0537-9
      19) Cell
      (T cell)
      FCM S. Yoshida, H. Nakagami, H. Hayashi, Y. Ikeda, J. Sun, A. Tenma, H. Tomioka, T. Kaawano, M. Shimamura, R. Morishita and H. Rakugi, "The CD153 vaccine is a senotherapeutic option for preventing the accumulation of senescent T cells in mice.", Nat. Commun., 2020, 11, (2482), doi:10.1038/s41467-020-16347-w
      20) Cell
      (PC12)
      Fluorescent microscope N. Wang, H. Wang, L. Li, Y. Li and R. Zhang, "β-Asarone Inhibits Amyloid-β by Promoting Autophagy in a Cell Model of Alzheimer's Disease.", Front Pharmacol., 2020, 10, 1529
      21) Cell
      (ARPE-19)
      Fluorescent microscope T. Yamazaki, H. Suzuki, S. Yamada, K. Ohshio, M. Sugamata, T. Yamada and Y. Morita, "Lactobacillus paracasei KW3110 Suppresses Inflammatory Stress-Induced Premature Cellular Senescence of Human Retinal Pigment Epithelium Cells and Reduces Ocular Disorders in Healthy Humans", Int J Mol Sci, 2020, 21(14), 5091