Welcome guest, Sign in

Home

X
加载中

Detection of senescent cells using a cytochemical assay was first described in 1995 (Dimri et al., 1995). The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity (Kurz et al., 2000). Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized in the lysosome. The enzymatic activity can be detected at the optimal pH 4.0, using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Miller, 1972). In comparison, upon senescence, the lysosomal mass is increased, leading to production of a higher level of β-galactosidase, termed senescence-associated β-galactosidase (SA-β-gal) (Kurz et al., 2000). The abundant senescence-associated enzyme is detectable over background despite the less favorable pH conditions (pH 6.0) (Dimri et al., 1995). The SA-β gal positive cells stain blue-green, which can be scored under bright-field microscopy. In this assay it is best to avoid over-confluency of the cells, or cells that have undergone too many passages, as these conditions can cause false positive results.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Senescence Associated β-galactosidase Staining

Cancer Biology > Cell death > Cell biology assays > Cell cycle
Authors: Michael Eccles
Michael EcclesAffiliation: Department of Pathology, University of Otago, Dunedin, New Zealand
For correspondence: michael.eccles@otago.ac.nz
Bio-protocol author page: a81
 and Caiyun Grace Li
Caiyun Grace LiAffiliation: Department of Pediatrics, Stanford University, Stanford, USA
Bio-protocol author page: a82
Vol 2, Iss 16, 8/20/2012, 18607 views, 7 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.247

[Abstract] Detection of senescent cells using a cytochemical assay was first described in 1995 (Dimri et al., 1995). The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity (Kurz et al., 2000). Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized in the lysosome. The enzymatic activity can be detected at the optimal pH 4.0, using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Miller, 1972). In comparison, upon senescence, the lysosomal mass is increased, leading to production of a higher level of β-galactosidase, termed senescence-associated β-galactosidase (SA-β-gal) (Kurz et al., 2000). The abundant senescence-associated enzyme is detectable over background despite the less favorable pH conditions (pH 6.0) (Dimri et al., 1995). The SA-β gal positive cells stain blue-green, which can be scored under bright-field microscopy. In this assay it is best to avoid over-confluency of the cells, or cells that have undergone too many passages, as these conditions can cause false positive results.

Keywords: Senescence, Beta-galactosidase, Colormetric

Materials and Reagents

  1. Paraformaldehyde (PFA) (Sigma-Aldrich)
  2. 5-bromo-4-chloro-3-indolyl β D-galactopyranoside (X-gal) (Sigma-Aldrich)
  3. Potassium ferrocyanide (Sigma-Aldrich, catalog number: B4252)
  4. Potassium ferricyanide (Sigma-Aldrich, catalog number: P9387)
  5. Phosphate buffered saline (PBS)
  6. Sodium hydroxide
  7. Dimethylformamide 
  8. Sodium chloride
  9. Magnesium chloride 
  10. Dibasic sodium phosphate
  11. Citric acid
  12. Sodium phosphate
  13. 4% paraformaldehyde (PFA) (see Recipes)
  14. Senescence associated β-galactosidase (SA-β-gal) staining solution (see Recipes)

Equipment

  1. Inverted microscope [e.g. Olympus 1 x 71 inverted microscope (Olympus)]
  2. 24-well plate
  3. p1000 pipette
  4. 37 °C incubator
  5. Hot plate

Procedure

  1. The manipulation of cells for the SA-β-gal staining assay may be performed in a 24-well plate format. 
  2. Prepare each sample in triplicate.
  3. At 120 h post-transfection or after the cell manipulation, aspirate the cell culture medium and wash the cells with PBS (500 μl per well) twice, using a p1000 pipette. 
  4. After the last rinse, replace the PBS with 250 μl of 4% PFA for fixation.
  5. Incubate the cells for 5 min at room temperature.
  6. Aspirate the 4% PFA and wash the cells two times for 5 min each at room temperature with gentle shaking with 500 μl PBS.
  7. Add 250 μl SA-β-gal staining solution to each well. 
  8. Incubate the cells in the dark in a 37 °C incubator. 
  9. Terminate the reactions when the cells are stained blue-green, as visualized under an inverted bright-field microscope. 
  10. To terminate the reaction, aspirate the staining solution and replace with distilled water.
  11. Wash the cells a second time in distilled water.
  12. After the last wash add 500 μl of distilled water to each well and observe the plate under an inverted bright-field microscope.
  13. Capture images of cells in each well using a 10x objective.
  14. Images may be printed for counting the total cell number, or count the stained cells on a computer monitor, which may give better distinction between the unstained and stained cells. 
  15. Represent the SA-β-gal positive cells as a percentage of the total cell number.
    Note: A trial may need to be carried out to determine the optimal length of incubation with SA-β-gal staining solution for each of the cell lines to be studied. Cells may be observed every 4 h during the first 12 h, and subsequently every 12 h.
    For example, the optimal incubation period in our hands was determined based on the visibility of the stained cells in test samples (e.g. PAX8siRNA treated samples) but not in the control samples (i.e. untreated sample or/and control siRNA treated sample). It is important to remember that the detection principle of this assay is based on the cellular abundance of the lysosomal β-galactosidase, which varies between cell lines. A table showing the length of optimal incubation time for several cell lines in our hands is as follows (Table 1).

Table 1. Example incubation times required for the appearance of the SA-β-gal activity in cell lines

Cell line Incubation time (h)
A498 (renal cell carcinoma) 24
786-O (renal cell carcinoma) 24
TK-10 (renal cell carcinoma) 12
K1 (thyroid carcinoma) 12


Figure 1A illustrates the appearance of increased SA-β-gal activity detected in response to PAX8 knockdown in four cell lines. Bright-field images show SA-β-gal positivecells in blue-green (insets). The colors of the images were inverted usingAdobe Photoshop (version 10.0) to aid the visibility of the positive (pink) cells. SA-β-gal positive cells were undetectable (or at a very low frequency) in control siRNA(SN) treated samples (highest detection was 3%, in TK-10 cells, Figure 1B). In comparison, adistinct elevation of SA-β-gal positive cells was observed in all PAX8 siRNA (S8) treated samples (highestdetection was 38%, in TK-10 cells).





Figure 1. Identification of senescent cells with the SA-β-gal staining assay. Cells treated with a control siRNA (SN), or PAX8 siRNA (S8) were assayed for SA-β-gal activity at 120 h post-siRNA treatment. A. Bright-field images are shown in the insets. These images were inverted using Adobe Photoshop to aid visibility of the positive cells (pink). The white arrows indicate precipitates from the staining solution. Magnification100x. B. Graph showing the percentage of positive cells (of the total cell number) in the treated samples.

Recipes

  1. 4% paraformaldehyde (PFA)
    To make 100 ml 4% PFA, dissolve 4 g PFA in 100 ml of PBS with continuous stirring on a hot plate (with the solution not exceeding 60 °C). Add 20 μl 1 M sodium hydroxide to dissolve the residual PFA. Aliquot and freeze at -20 °C for long-term storage.
  2. Senescence associated β-galactosidase (SA-β-gal) staining solution
    Firstly, prepare the following stock solutions.
    1. To prepare 10% X-gal in dimethylformamide (DMF) dissolve 1 g X-gal in 10 ml DMF. Store the stock X-gal solution at -20 °C.
    2. 400 mM citric acid/sodium phosphate solution add 36.85 ml 0.1 M citric acid to 63.15 ml 0.2 M dibasic sodium phosphate. Verify the pH and adjust to pH 6.0 with 0.1 M citric acid, if necessary.
    3. 0.5 M potassium ferrocyanide and 0.5 M potassium ferricyanide, Store stock solutions of potassium ferrocyanide and potassium ferricyanide in the dark at 4 °C.
    To prepare the SA-β-gal staining solution, make appropriate dilutions of stock solutions with water to give a solution containing 0.1% X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM Sodium chloride, and 2 mM Magnesium chloride in 40 mM citric acid/sodium phosphate solution, pH 6.0.

Acknowledgments

This protocol is based on the same protocol as published in Li et al. (2011). MRE wishes to acknowledge support from the University of Otago Leading Thinkers Advancement Campaign, and the New Zealand Institute for Cancer Research Trust. CGL wishes to acknowledge scholarship support from the Health Research Council, the University of Otago International Fees Scholarship and Postgraduate Publishing Bursary and the Dunedin School of Medicine Finishing Your PhD Scholarship. Research grants supporting the implementation of this protocol were from the Health Research Council of New Zealand and the University of Otago Faculty of Medicine Trust Fund.

References

  1. Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., Pereira-Smith, O. and et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92(20): 9363-9367.
  2. Kurz, D. J., Decary, S., Hong, Y. and Erusalimsky, J. D. (2000). Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 113 (Pt 20): 3613-3622.
  3. Li, C. G., Nyman, J. E., Braithwaite, A. W. and Eccles, M. R. (2011). PAX8 promotes tumor cell growth by transcriptionally regulating E2F1 and stabilizing RB protein. Oncogene 30(48): 4824-4834. 
  4. Miller, J. H. (1972). Experiments in molecular genetics. Cold Spring Harbor Laboratory xvi, 466 p.pp.


How to cite: Eccles, M. and Li, C. G. (2012). Senescence Associated β-galactosidase Staining. Bio-protocol 2(16): e247. DOI: 10.21769/BioProtoc.247; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
10/13/2015 7:44:09 PM  

Nada Alshehri
Forsyth institute

i posted my question below, about crystals

10/22/2015 3:08:12 PM  

Bio-protocol Editorial Team
bio-protocol.org

Hi Nada,

Your question about crystals could not be found. Would you please re-post it? Thanks.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

4/2/2015 7:25:19 PM  

Parul Rai
UC Berkeley

Hi I have 2 questions
1. For how many hours do I incubate hematopoietic stem cells after staining them?
2. I sort my stem cells directly onto a slide and wait for about 45 min to let them adhere to the poly-lysine slide prior to fixing them. Unfortunately I have lost the cells while attempting to wash them after fixation and was wondering whether it is possible to stain them immediately after fixation without washing the cells ?

Thanks

4/2/2015 7:56:46 PM  

Caiyun Li (Author)
Department of Pediatrics,Stanford University

Hi Paul,
Here is the answer -

Q1: We do not have experience with stem cells. I would suggest you to monitor the color development anytime from 6h to 48h (or even longer) incubation, until you see blue colored cells.

Q2: Residual fixative (PFA) might affect staining and reproducibility. I would suggest to wash at least twice before proceed. However, I would suggest to coat dishes/slides with different reagents (fibronectin, collagen, or gelatin) to reduce detachment of the cells.

Hope this helps.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

2/27/2014 6:01:00 AM  

S Gnanadesikan
University of Glasgow

carried without CO2**?

2/27/2014 8:21:15 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

The incubator was a normal dry incubator – no CO2 control.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

2/27/2014 6:00:22 AM  

S Gnanadesikan
University of Glasgow

Why is the incubation carried out in the incubator with CO2?

2/27/2014 8:20:54 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

The incubator was a normal dry incubator – no CO2 control.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

2/27/2014 5:54:02 AM  

S Gnanadesikan
University of Glasgow

Why is the incubation carried out in the dark?any specific reasons?

2/27/2014 8:21:44 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

It was in dark because X-gal the substrate is light sensitive.

2/28/2014 2:55:12 AM  

S Gnanadesikan
University of Glasgow

Thank you for the answer..

3/4/2014 3:22:06 AM  

S Gnanadesikan
University of Glasgow

May I know what exactly is the staining solution A and staining solution B that is used for preparation of SA-ß-gal solution?

3/4/2014 12:02:55 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

To see what exactly the staining solution is see under "Recipes" item 2. The correspondent will see there is not exactly a staining solution A nor staining solution B. There are three stock solutions to prepare, before proceeding to make the staining solution.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

11/26/2012 2:50:35 PM  

I noticed several salt crystals post SA-bGal staining, which do not seem to dissolve after washing. These crystals interfere during counting and also while taking pictures. Is there anyway to troubleshoot this problem?

Thanks,
Vijay

11/28/2012 1:42:49 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

We have also seen these crystals before. However, we have always managed to take images without excessive interference from the crystals. Our only suggestion is to do as many washes with water after the staining as necessary.

10/13/2015 7:39:44 PM  

Nada Alshehri
Forsyth institute

My first question is about what type pf water should i use to remove the crystals?

The number of my crystals is so many and attached to the flask surface that i can't read the results properly. so how do I dissolve them


my second question is about false positive staining
I have observed blue stained cells in control sample of young , cells weren't over confluent , is it normal to have some blue staining in control young cells
my cells were Periodontal Ligament fibroblasts, and they were stained two starting from passage 2
Thanks a lot
Nada

       

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

9/5/2012 1:33:55 AM  

To Whom It May Concern,

One question for the Beta-gal assay: will the stained cell number change (/increase) along with the incubation time? My experience is that I got ~40% blue cells at the 6th hour, and obtain ~90% blue cells at the 24th hour (they were in the same cell pool, and I used HUVEC, passage of 3. We did not wash out the stain solution until the last observation at 24th hour)?! Should I pick up 6 or 24 hour of incubation for the experiment?

Thank you for the answer.

Bill

9/5/2012 1:53:35 PM  

Michael Eccles (Author)
Department of Pathology,University of Otago

answer:
in our experience, different cell lines require different incubation times. If HUVEC naturally have high endogenous galactosidase, the pH of the incubation buffer will be critical. If you see blue cells ONLY in the treatment known to induce senescence, then 6 hours might be better, assuming this time point is in the linear range of the senescence development. Having a positive control will be important (e.g. using very late passage cells that have stopped dividing).

hope this helps.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook