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Senescence Associated β-galactosidase Staining
衰老相关β半乳糖苷酶的染色

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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(衰老), Beta-galactosidase(β-半乳糖苷), Colormetric(比色)

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.

简介

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.

关键字:衰老, β-半乳糖苷, 比色

材料和试剂

  1. 多聚甲醛(PFA)(Sigma-Aldrich)
  2. 5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷(X-gal)(Sigma-Aldrich)
  3. 亚铁氰化钾(Sigma-Aldrich,目录号:B4252)
  4. 铁氰化钾(Sigma-Aldrich,目录号:P9387)
  5. 磷酸盐缓冲盐水(PBS)
  6. 氢氧化钠
  7. 二甲基甲酰胺
  8. 氯化钠
  9. 氯化镁
  10. 磷酸二钠
  11. 柠檬酸
  12. 磷酸钠
  13. 4%多聚甲醛(PFA)(参见配方)
  14. 衰老相关的β-半乳糖苷酶(SA-β-gal)染色溶液(参见配方)

设备

  1. 倒置显微镜[ Olympus 1 x 71倒置显微镜(奥林巴斯)]
  2. 24孔板
  3. p1000移液器
  4. 37℃孵育器
  5. 热板

程序

  1. 用于SA-β-gal染色测定的细胞操作可以在24孔板格式中进行。
  2. 准备每个样品一式三份。
  3. 在转染后120小时或细胞操作后,吸出细胞培养基,用PBS(500μl/孔)洗涤细胞两次,使用p1000移液管。
  4. 最后一次冲洗后,用250μl的4%PFA替换PBS固定。
  5. 在室温下孵育细胞5分钟。
  6. 吸出4%PFA,洗涤细胞两次,每次5分钟,在室温下与500微升PBS轻轻摇动。
  7. 每孔加入250μlSA-β-gal染色溶液。
  8. 孵育细胞在黑暗中37℃的孵化器。
  9. 当细胞染成蓝绿色时,终止反应,如在倒置明场显微镜下可见。
  10. 要终止反应,请吸出染色溶液并更换蒸馏水。
  11. 在蒸馏水中第二次洗涤细胞。
  12. 最后一次洗涤后,向每个孔中加入500μl蒸馏水,并在倒置明场显微镜下观察板。
  13. 使用10x物镜捕获每个孔中的细胞的图像。
  14. 可以打印图像以计数总细胞数目,或在计算机监视器上计数染色的细胞,这可以在未染色和染色的细胞之间给出更好的区别。
  15. 代表SA-β-gal阳性细胞占总细胞数的百分比。
    注意:可能需要进行试验,以确定每个待研究的细胞系与SA-β-gal染色溶液孵育的最佳长度。在前12小时期间每4小时观察细胞,随后每12小时观察细胞。例如, PAX8siRNA处理的样品),而不是在对照样品( 未处理的样品或/和对照siRNA处理的样品)中。重要的是记住,该测定的检测原理是基于溶酶体β-半乳糖苷酶的细胞丰度,其在细胞系之间变化。显示我们手中的几种细胞系的最佳孵育时间的长度的表如下(表1)。

表1. 细胞系中SA-β-gal活性出现所需的孵育时间示例

单元格行 孵育时间(h)
A498(肾细胞癌) 24
786-O(肾细胞癌) 24
TK-10(肾细胞癌) 12
K1(甲状腺癌) 12


图1A图示了在四种细胞系中响应于PAX8敲低检测到的增加的SA-β-gal活性的外观。明场图像显示蓝绿色SA-β-gal阳性细胞(插图)。使用Adobe Photoshop(版本10.0)颠倒图像的颜色以帮助阳性(粉红色)细胞的可见性。 SA-β-gal阳性细胞在对照siRNA(SN)处理的样品(在TK-10细胞中,最高检测为3%,图1B)中检测不到(或以非常低的频率)。相比之下,在所有PAX8siRNA(S8)处理的样品中观察到SA-β-gal阳性细胞的不明显升高(在TK-10细胞中最高检测为38%)。





图1。 用SA-β-gal染色测定法鉴定衰老细胞。 在siRNA处理后120小时测定用对照siRNA(SN)或PAX8 siRNA(S8)处理的细胞的SA-β-gal活性。 A.亮场图像显示在插图中。使用Adobe Photoshop反转这些图像以帮助阳性细胞(粉红色)的可见性。白色箭头表示来自染色溶液的沉淀。放大100倍。 B.显示处理样品中阳性细胞(总细胞数)百分比的图。

食谱

  1. 4%多聚甲醛(PFA)
    为了制备100ml 4%PFA,在连续搅拌下在热板上(溶液不超过60℃)溶解4g PFA在100ml PBS中。加入20μl1M氢氧化钠溶解残余的PFA。等分并在-20°C冷冻 长期储存。
  2. 衰老相关的β-半乳糖苷酶(SA-β-gal)染色溶液
    首先,准备以下储备溶液。
    1. 为了在二甲基甲酰胺(DMF)中制备10%X-gal,将10ml X-gal溶解在10ml DMF中。将储备的X-gal溶液储存在-20°C。
    2. 400mM柠檬酸/磷酸钠溶液中加入36.85ml 0.1M柠檬酸至63.15ml 0.2M磷酸氢二钠。如果需要,用0.1M柠檬酸验证pH并调节至pH6.0。
    3. 0.5M亚铁氰化钾和0.5M铁氰化钾,在4℃黑暗中储存亚铁氰化钾和铁氰化钾的储备溶液。
    为了制备SA-β-gal染色溶液,用水适当稀释储备溶液,得到含有0.1%X-gal,5mM亚铁氰化钾,5mM铁氰化钾,150mM氯化钠和2mM氯化镁的溶液在40mM柠檬酸/磷酸钠溶液,pH 6.0中。

致谢

该协议基于与Li等人(2011)中公布的相同的协议。 MRE希望承认奥塔哥大学领导思想家进步运动和新西兰癌症研究信托研究所的支持。 CGL希望承认健康研究委员会,奥塔哥国际费用奖学金和研究生出版奖学金和达尼丁医学院完成您的博士奖学金的奖学金支持。支持该协议实施的研究补助金来自新西兰健康研究委员会和奥塔哥大学医学院信托基金。

参考文献

  1. Dimri,GP,Lee,X.,Basile,G.,Acosta,M.,Scott,G.,Roskelley,C.,Medrano,EE,Linskens,M.,Rubelj,I.,Pereira-Smith, 等。 (1995)。 在体内识别文化和衰老皮肤中的衰老人类细胞的生物标志物。 Proc Natl Acad Sci USA 92(20):9363-9367。
  2. Kurz,D.J.,Decary,S.,Hong,Y。和Erusalimsky,J.D。(2000)。 衰老相关(β) - 半乳糖苷酶反映溶酶体质量的增加 在人内皮细胞的复制性老化期间。 J Cell Sci 111(Pt 20):3613-3622。
  3. Li,C.G.,Nyman,J.E.,Braithwaite,A.W.and Eccles,M.R。(2011)。 PAX8通过转录调节E2F1和稳定RB蛋白来促进肿瘤细胞生长。 Oncogene 30(48):4824-4834。
  4. Miller,J.H。(1972)。 分子遗传学实验。 Cold Spring Harbor Laboratory xvi,466 p.pp.
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引用:Eccles, M. and Li, C. G. (2012). Senescence Associated β-galactosidase Staining. Bio-protocol 2(16): e247. DOI: 10.21769/BioProtoc.247.
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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
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
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
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
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
Department of Pathology, University of Otago, New Zealand

Thanks very much for this.

4/19/2017 8:57:11 PM


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.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
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 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
University of Glasgow
carried without CO2**?
2/27/2014 6:01:00 AM Reply
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
University of Glasgow
Why is the incubation carried out in the incubator with CO2?
2/27/2014 6:00:22 AM Reply
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
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
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
University of Glasgow

Thank you for the answer..

2/28/2014 2:55:12 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 3:22:06 AM


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
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
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
Forsyth institute

10/13/2015 7:42:45 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
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