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Senescence Associated β-galactosidase Staining    

Michael EcclesMichael Eccles Caiyun Grace LiCaiyun Grace Li
DOI:   10.21769/BioProtoc.247
Published:   Vol 2, Iss 16, August 20, 2012
Cancer Biology > Cell death > Cell biology assays > Cell cycle
Cell Biology > Cell viability > Cell death
Cell Biology > Cell staining > Whole cell
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In this protocol

Original research article

A brief version of this protocol appeared in:
Dec 2011

 

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.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
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.
Q&A

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Rabab Mahmoud El Genedy
Rabab Mahmoud El Genedy
University of Sheffield
Thank You for sharing this protocol : I have 4 questions
1- How could I determine the PH in my reaction? I do the same volumes mentioned in the kit which differ according to the plate size (usually do 10 cm petri dish , without assessing the PH) I didn`t get proper greenish bluish staining , usually more brownish , I incubate them for 24 hours in dry 37 degree incubator without co2 as recommended.

2- My treatment is UV irradiation , do you have any idea how long should I keep cells after UV ttt to be able to attain senescence? in some paper they left them up to 12 days which is hard for my cells to be left all this time without need to be passaged?

3- What is the recommended initial number or confluency of the flask prior to ttt if you intend to keep cells 5 days for ex. before fixation and staining?

4- I use the fixative solution that come with the kit and it is mentioned to be kept for 12-15 min. before washing, Is this different than paraformaldehyde 4% you are using?
1/7/2020 6:05:08 AM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

1- How could I determine the PH in my reaction? I do the same volumes mentioned in the kit which differ according to the plate size (usually do 10 cm petri dish , without assessing the PH) I didn`t get proper greenish bluish staining , usually more brownish , I incubate them for 24 hours in dry 37 degree incubator without co2 as recommended.

If you are using a kit, i would strictly follow the kit instructions. I expect most reagents would be similar to our protocol. I have never seen any brownish staining in my experiments, it would be helpful to post your photo here.

2- My treatment is UV irradiation , do you have any idea how long should I keep cells after UV ttt to be able to attain senescence? in some paper they left them up to 12 days which is hard for my cells to be left all this time without need to be passaged?

I would not expect senescence to be induced right after UV irradiation. It would depend if the cells are able to repair the UV damage in the first place. If you predict to see replicative senescence, it is normal that it would take 12 days or more, or multiple passages until the cells are not able to replicate anymore. In our case, our manipulation permanently arrested cells at G1 phase, that's why it took only a few days to see the senescence phenotypes.

3- What is the recommended initial number or confluency of the flask prior to ttt if you intend to keep cells 5 days for ex. before fixation and staining?

It depends very much on the doubling time of the cells and how tolerance the cells are to seeding at very low confluence. If your cells are replicating for 5 days (without any growth arrest), then it is unlikely that the cells would be undergoing senescence in the first place. If your treatment is supposed to slow down growth but does not induce cell death, you could try to seed 10-15% of the growth surface area first.

4- I use the fixative solution that come with the kit and it is mentioned to be kept for 12-15 min. before washing, Is this different than paraformaldehyde 4% you are using?

Again, I am not familiar with the kit. However, PFA is one the most common fixatives. I would assume it could be the same or similar.

2/18/2020 6:01:07 PM

NEHA JAIN
NEHA JAIN
National Institute of technology , Rourkela
I have done senescence staining before with the above protocol, now my staining does not come. What can be the reason. I am using new X-Gal lot or can the problem comes for potassium ferrocyanide or potassium ferricyanide powder being exposed to light.
2/8/2019 3:23:14 AM Reply
anamika datta
anamika datta
kumamoto university

Dear Neha
You did not mentioned about your cell type. From my experience, X-gal staining needs to be optimize for specific cell type.You may check the whether the working solution has been prepared accordingly. Try to avoid repeated freeze-thawing the solutions. I usually aliquot working stock to least required volumes to avoid this. You may optimize your working concentration molarity, incubation time as per your cell type. Be careful for fixation the cells, prolonged fixation may damage the cell. Try to take stained cells picture as soon as possible, sometimes this is also responsible for bad staining results. Last but not the least, expertise of the microscope is a key to get good staining results.
Best wishes

2/8/2019 4:16:14 AM

Sivilay XAY
Sivilay XAY
Yeungnam university
Dear Michael and Grace,

Thank you very much for your wonderful protocol.
I did performed the SA-β-gal staining in MRC-5, but MRC-5 was very sensitive to wahsing with PBS. I mean my MRC-5 is fall out after wasing 2 time with PBS.
I did skip the washing step before fixing, my cell was ok but SA-β-gal staining was not work.
Could you please give me any suggestion?
4/17/2018 10:26:24 PM Reply
neil Fisher
neil Fisher
University of Basel
Hi there. Do you know whether incubation at a slightly lower temperature (say, 35°C) negatively affects staining, or whether you simply have to bake the samples for longer? cheers Neil
11/1/2017 9:05:58 AM Reply
Nathan Tang
Nathan Tang
Bergen County Academies
Why do crystals form? Do they form in the staining solution or when the staining solution was applied? Also, why do you have to wait 5 days after you have induced senescence in order to stain? Does that mean that the cells go through 5 divisions and then become senescent? Because if the cells became senescent immediately, shouldn't they be stained as soon as possible?
7/11/2017 10:48:47 AM Reply
Kitson Liew
Kitson Liew
Institute for Medical Research, Kuala Lumpur, Malaysia
Dear Michael and Grace,

Thank you very much for sharing this wonderful protocol.

May I know if I can keep the fixed cells in PBS at 4 C as a pause step after fixing and washing twice with PBS? If yes, would there be a limit to how long I can keep the fixed cells in such a way prior to staining?

I am worried that if I am working with a large number of plates, I am not able to stain all of them in one day.

Thank you once again,

Kitson
5/23/2017 9:03:23 PM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

We stained cells immediately after fixation. Please note that the fixation time is short, only 5min. I would suggest to process the cells as promptly as you can. If you have to, I would suggest to store fixed cells in PBS, seal it with parafilm and keep the samples at 4 degree Celsius. I would process the cells within 2-3 days, PBS salt crystals would eventually form in PBS and could affect staining/imaging. I would run a trial to compare if there is any staining positivity with the different storage period (immediate processing vs. after 2-3 days).

5/25/2017 10:04:27 PM

Kitson Liew
Kitson Liew
Institute for Medical Research, Kuala Lumpur, Malaysia

Thank you very much, Michael for your reply!

May I know if immunofluorescence (IF) staining can still be carried out on fixed cells previously stained for SAbeta-gal activity?

If yes, what would be the recommended procedures for (IF) after the SAbeta-gal staining?

Many thanks once again.

5/26/2017 12:24:25 AM

catalina rubio
catalina rubio
University of tolima
Hi! I'm very grateful with you for share this protocol, i've been testing it with different Line cells like T98g and Stem cells.

I'm agree with you this assay it is best to avoid over-confluency of the cells, and will have a better staining , and at the beginning I had some problems with the crystals too, then solve this by performing several washes with PBS, i calculate the time for different cells, T98g It takes 12 hours and the stem cells take about 48 hours to be stained.

I add two photos, the first is the test in stem cells and second is the test in T98g.


I apologize for my English and thank you again for sharing your protocol. It has been very helpful for me.

4/19/2017 8:11:59 PM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

Thanks very much for this.

4/19/2017 8:57:11 PM

yan huang
yan huang
Sun Yat-sen university

I tried to use ethanol to remove the crystals and it works, 3-5 hours enough, hope to help you.

12/5/2019 6:57:54 PM

Nada Alshehri
Nada Alshehri
Forsyth institute
i posted my question below, about crystals
10/13/2015 7:44:09 PM Reply
Bio-protocol Editorial Team
Bio-protocol Editorial Team
bio-protocol.org

Hi Nada,

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

10/22/2015 3:08:12 PM

Parul Rai
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 7:25:19 PM Reply
Caiyun Grace Li
Caiyun Grace Li
Department of Pediatrics, Stanford University, USA

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.

4/2/2015 7:56:46 PM

S Gnanadesikan
S Gnanadesikan
University of Glasgow
carried without CO2**?
2/27/2014 6:01:00 AM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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

2/27/2014 8:21:15 PM

S Gnanadesikan
S Gnanadesikan
University of Glasgow
Why is the incubation carried out in the incubator with CO2?
2/27/2014 6:00:22 AM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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

2/27/2014 8:20:54 PM

S Gnanadesikan
S Gnanadesikan
University of Glasgow
Why is the incubation carried out in the dark?any specific reasons?
2/27/2014 5:54:02 AM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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

2/27/2014 8:21:44 PM

S Gnanadesikan
S Gnanadesikan
University of Glasgow

Thank you for the answer..

2/28/2014 2:55:12 AM

S Gnanadesikan
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 3:22:06 AM

Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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.

3/4/2014 12:02:55 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
11/26/2012 2:50:35 PM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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.

11/28/2012 1:42:49 PM

Nada Alshehri
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

10/13/2015 7:39:44 PM

Nada Alshehri
Nada Alshehri
Forsyth institute

10/13/2015 7:42:45 PM

yan huang
yan huang
Sun Yat-sen university

I tried to use ethanol to remove the crystals and it works, 3-5 hours enough, hope to help you.

12/5/2019 6:58:45 PM

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 1:33:55 AM Reply
Michael Eccles
Michael Eccles
Department of Pathology, University of Otago, New Zealand

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.

9/5/2012 1:53:35 PM

chao fan
chao fan
重庆市大坪医院心内科

Dear Bill， It's real surprising and even exciting to see your writing here，I‘m doing the same experiment with HUVEC，it's real hard to get the perfect result because all the cells became blue after 24h staining ，maybe I should shorten the time as you advised. Thanks very much！May I got your email？I hope to ask more question from you .

7/10/2019 11:45:19 PM

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