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
Equipment
Procedure
Table 1. Example incubation times required for the appearance of the SA-β-gal activity in cell lines
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
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
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Fixation is performed to keep the cell/tissue sample to be in its original form and characteristics, so that during the experiment the sample can stay in a permanent condition. Fixation agents works in different ways, for example covalent cross-links between molecules can be initiated by Paraformaldehyde, which effectively attached them together to form an insoluble meshwork. For immunostaining, the staining reagent need to permeabilize into the cells. But if the cells are not fixed, the structure of the cell shape may change which may cause the staining reagent to be diffused away before the incubation has been finished. Also, the sample should be in small volume to allow better access of the fixation solution and the fixation solution volume should be at least twenty times more than the specimen volume. After fixation, the sample should be washed to remove the fixation traces completely unless it may hinder proper staining of the sample. Before fixation, please check the cell type and size to decide the type and volume of fixation solution. Regards.
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.
Dear NehaYou 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
Crystals develop if the staining solution evaporates from the wells.To prevent this, seal your plates with ParaFilm.
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).
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.
Thanks very much for this.
I tried to use ethanol to remove the crystals and it works, 3-5 hours enough, hope to help you.
Hi Nada,Your question about crystals could not be found. Would you please re-post it? Thanks.
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.
The incubator was a normal dry incubator – no CO2 control.
It was in dark because X-gal the substrate is light sensitive.
Thank you for the answer..
May I know what exactly is the staining solution A and staining solution B that is used for preparation of SA-ß-gal solution?
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.
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.
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 themmy second question is about false positive stainingI 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 cellsmy cells were Periodontal Ligament fibroblasts, and they were stained two starting from passage 2Thanks a lotNada
what is the concentration of ethanol you used？thank you
Hi Yan,Did you use 70% ethanol or 100% ethanol to wash?
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.
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 .
