Several methods were gradually developed for the detection of DNA synthesis in cell nuclei. Presently, the approach based on the use of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by click reaction increasingly dominates among them. Another widely used method is the detection of DNA replication using 5-bromo-2′-deoxyuridine (BrdU) or eventually other thymidine analogues — iodo-2′-deoxyuridine (IdU) or 5-chloro-2′-deoxyuridine (CldU) — by means of specific antibodies. Additional methods, like the use of biotinylated nucleotides, require specific steps for their introduction into cells and are not commonly used. If there is need for such approach, you can use e.g. this protocol.
In the case of EdU, the most important problems are connected with its high cytotoxicity. Therefore, EdU is not suitable for long-term studies. Although another ethynyl analogue, namely 5-ethynyl-2′-deoxycytidine (EdC) was previously suggested as a low toxic substitution for EdU, our previous study clearly showed that EdC is not incorporated into DNA (Ligasová et al., 2016). Instead EdC is first deaminated to EdU and in this form is incorporated into DNA. The major part of EdC toxicity results from its transformation into EdU and the incorporation of EdU into DNA. Thus, the lower toxicity of EdC is accompanied by the lower replicational signal, as only a fraction of EdC is transformed to EdU during the labelling pulse.
In addition, the click reaction by means of copper ions results in the formation of oxygen radicals, which cause DNA and RNA damage and can impair the fluorescence of fluorescent proteins such as GFP as well. The production of oxygen radicals can be minimized by the use of some additives; however, their presence can result in the lowering of the EdU signal and increases the costs of such an approach.
Probably the most important limitation of 5-halogen analogues of thymidine (BrdU, IdU and CldU) is that they are commonly inaccessible in the double-stranded DNA for reaction with the specific antibodies. In this case, special approaches are required to make them accessible. The most used methods are based on hydrochloric acid (HCl) or sodium hydroxide or DNase I. In addition, protocols based on monovalent copper ions and nucleases have been developed. However, the use of the mentioned approaches can result in considerable changes of the cell structure including the strong and uncontrolled destruction of DNA, RNA or proteins. It can also result in the loss of cells during sample preparation for the cell cycle analysis by flow cytometry. In addition, the signal depends on the antibody used.
Moreover, the signal depends on the antibody used. As there is a high number of monoclonal antibody clones raised against BrdU available on the market, the choice of the suitable antibody can be a pretty tough task. In this respect, we tested the reactivity of ten different anti-BrdU antibody clones with three oligonucleotides containing BrdU alternatively at 5′ or 3′ end or in the central part of the oligonucleotide chain. We showed that every clone exhibited a specific combination of the three affinity constants providing a characteristic pattern for every antibody (its fingerprint, Ligasová et al., 2015). The simultaneously performed analysis of the BrdU-derived signal in replicated cells using these antibodies and four different protocols of BrdU detection showed that the analysis of the fingerprints can serve as a reliable guide for the estimation of the reactivity of the anti-BrdU clone with the incorporated BrdU in fixed and permeabilized cells.
Interestingly, only two tested clones were suitable for the detection of BrdU in all the tested protocols. Unfortunately, the least cell damaging approaches based on the use of DNase I exhibit a high dependency on the antibody used.
Recently we have shown that there are two ways to substantially improve the signal when DNase I treatment is used and to make this procedure less antibody dependent. It involves the use of i) exonuclease III simultaneously with DNase I and most importantly, ii) the post-fixation of samples with formaldehyde after BrdU detection (Ligasová et al., 2017).
Although the post-fixation step can be partially overcome with the fast reaction with the secondary antibody, the formaldehyde post-fixation step provides a better signal/background ratio and, e.g. in the case of samples prepared for flow cytometry, it is the only variant how to quickly stabilize the BrdU-antibody complex. This also clearly showed the importance of the highly specific antibody as only in the case of antibodies exhibiting high specificity and low affinity can the fast stabilization substantially improve results.
It is obvious that it is not easy to find and choose the correct detection protocol for the specific experimental requirements. Here we provide a brief guide to simplify the selection. There are several aspects that have to be taken into account when someone is looking for an appropriate protocol.
1. Incorporation rate. EdU is much less willingly incorporated into DNA than thymidine or BrdU. As the thymidine pool exhibits a much higher negative effect on EdU incorporation than BrdU incorporation, the minimal effective concentration of EdU should usually be higher than the concentration of BrdU. This is also the reason why synchronization protocols based on the use of elevated concentrations of thymidine are not compatible with EdU incorporation – the high intracellular concentrations of thymidine compete with EdU and stop its incorporation into DNA for a long time. In this respect, in double labelling experiments with BrdU and EdU, the EdU pulse should precede the BrdU pulse. It especially concerns cases if there is no or only a very short chase period between pulses or thymidine is used during the chase to stop the incorporation of the first analogue. The slower incorporation rate of EdU than of BrdU also means that EdU is not suitable for short pulses lasting several minutes. In this case, BrdU is definitely superior do EdU.
2. Cross-talk between EdU-bound azido fluorochrome and DNA stains as a result of Fluorescence Resonance Energy Transfer (FRET). This aspect should be taken into account as well, although it is only seldom mentioned. For instance, if FAM-azide (FAM – carboxyfluorescein) is used for the detection of incorporated EdU and DAPI is used for the staining of the overall DNA, the significant decrease of the DAPI signal is observed. This drop in the DAPI signal increases with the length of the EdU incorporation. It can significantly change, e.g., the histograms of DAPI signals used for the analysis of the cell cycle. If FAM-azide and propidium iodide are used instead of DAPI, the FAM signal decreases significantly. The decrease is insensitive to the length of the EdU pulse. Such cross-talk is not observed in the case of BrdU detection as the fluorochrome is bound to the antibody and not directly to DNA. It should also be mentioned that the incorporation of BrdU or EdU by itself decreases the fluorescence signal of Hoechst stains bound to DNA. No such effect is observed if DAPI or propidium iodide are used.
3. Toxicity. As EdU is much more toxic than BrdU, it is not suitable for studies based on long pulses or pulse-chase experiments. The judgement of what a long experiment is will depend on the length of the cell cycle, the purpose of study and cell sensitivity. In the case of highly sensitive cell lines with a cell cycle around 12 hours and studies focused on the cell cycle progression two hours can be too long a time. In this respect, 4- to 6- hour experiments for HeLa cells with the doubling time around 24 hours can be performed for such studies, as only subtle lowering of the progression of cells during the cell cycle was observed. EdU incorporation can result in DNA double strand breaks and in cell death (Ligasová et al., 2015).
4. Inhibition of thymidylate synthase. It should be also mentioned that EdU exhibits an inhibitory effect on thymidylate synthase (see e.g. Ligasová et al., 2015), although much lower than, e.g., FdU. No such effect was described for BrdU and, therefore, EdU is not suitable in studies focused on the analysis of pathways of nucleoside and nucleotide synthesis. As the incorporation of EdU is accompanied by the induction of DNA lesions, its use for studies focused on DNA repair should also be judged carefully.
5. Simultaneous detection of replication and other cellular components or fluorescent proteins. Although EdU is commonly recommended in the case of the simultaneous detection of replication and cellular components, some BrdU detection protocols provide similar or in some cases even better results. In the case of the simultaneous detection of fluorescent proteins such as GFP, BrdU is definitely a better choice than EdU.
6. The speed of the approach. The method based on EdU is very fast as compared to the traditional BrdU detection protocols. The use of DNA stains prolongs the procedure based on EdU a little bit as DNA stains should be added after the reaction with EdU. Although we tested the addition of DAPI to the reaction mixture containing copper ions, it usually resulted in a lower DAPI signal. In the case of BrdU, DNA dyes can be present in the mixture of primary anti-BrdU or secondary antibody without any significant effect on the DNA signal. Despite that, EdU detection is still a faster protocol as DNA staining can be performed very quickly. The length of the approach based on EdU and BrdU can be similar if the simultaneous detection of other cellular components by antibodies is performed. In this case, EdU detection is an individual step, while the detection of BrdU and cellular components can be performed at the same time.
7. Anti-BrdU antibodies. There are a number of different anti-BrdU antibody clones on the market. All of them have a different affinity to incorporated BrdU. Therefore, it is necessary to choose the right one. In this respect, most of the described protocols also contain data about the recommended antibody.
8. Detection of mitochondrial replication. In this case, BrdU is superior to EdU. The detection of mitochondrial replication suffers from the fact that the nuclear signal is usually much higher than the mitochondrial signal. Therefore, the nuclear signal can effectively obscure the mitochondrial signal. As the revelation step is necessary to detect BrdU in DNA, it provides the possibility to modulate this step in such way that the mitochondrial replication is detected preferentially. No such possibility is available in the case of EdU.
9. The price of the whole procedure. In this respect, BrdU is much cheaper than EdU. In addition, many people use the kits for the detection of EdU and expensive azido-dyes. Therefore, BrdU seems to be more convenient for the detection of replicational activity in cells if a large number of samples should be processed although the price of anti-BrdU antibodies is not negligible. Surprisingly, if you buy azido conjugates of FAM or TAMRA (carboxytetramethylrhodamine) fluorochromes and spend a half an hour preparing the solutions of sodium ascorbate and copper sulfate as described here, you have an inexpensive solution for the detection of replication activity as well.
The following table summarizes all the above-mentioned aspects including some recommended protocols.
|The labelling pulse is shorter than 20 minutes||–
The minimal time necessary for the successful detection of EdU depends on the cell line used and the concentration of EdU as different cell lines incorporate EdU with different efficiency (citace). The time has to be tested. We recommend comparing the number of EdU-labelled cells with BrdU-labelled cells. The differences in the ability of individual cells to incorporate EdU can result in the impression, that cells incorporate EdU well. On the other hand, the labelled cells can represent only a fraction of the replicating cells. It inevitably leads to an underestimation of the number of replicating cells.
A typical concentration of BrdU is 10 µM for most cell lines.
A 5-10-minute labelling pulse usually provides a sufficiently high signal to clearly distinguish replicating and non-replicating cells using cell sorters or by image cytometry software e.g. Cell Profiler.
|The labelling pulse is longer than 20 minutes and shorter than the time inducing the toxic effect of EdU||+++
The decision of what labelling time is too long will depend on the purpose of the study. E.g. if the only purpose is to estimate the number of cycling cells, the times that do not result in cell death can be used. In this respect, e.g. a 12-hour incubation time is OK for HeLa cells. On the other hand, if the effect on the cell cycle is studied and a sensitive cell line is used, already a 2-hour incubation of cells with EdU can result in a significant decrease of the speed of cell transition during the S-phase.
Although BrdU is toxic for cells as well, its toxicity is much lower than in the case of EdU.
The longer labelling time means that usually a higher dilution of anti-BrdU antibody can be used.
|Long labelling pulse or chase after the pulse are necessary (usually several hours up to several days).||–
EdU incorporation results in DNA damage and cell death. This feature cannot be overcome by the decrease of the EdU concentration as a decrease of concentration to non-toxic levels simultaneously leads to a low or no replication signal.
If the labelling pulse or chase after the labelling pulse lasts several hours or days, the toxicity of BrdU should be analyzed first. A long BrdU labelling pulse also means that most protocols used for BrdU detection can be successfully used.
Recommended protocol: 4.
This protocol preferentially reveals BrdU in mitochondrial DNA.
|Simple analysis of cell cycle using FACS and 30-minute labelling pulse||+++
Recommended protocol: 3a
Recommended protocol: 1a
|Simple analysis of cell cycle using image cytometry and 30-minute labelling pulse||+++
Recommended protocol: 3
|Simultaneous localization of other cellular components using specific antibody||++
The lower recommendation rank results from the fact that a higher number of centrifugation steps is necessary as compared to BrdU detection in the case of FACS applications.
The detection using specific antibodies is performed simultaneously with the detection of BrdU.
|Simultaneous analysis of fluorescent proteins||–||+++
|Synchronization protocol based on elevated concentration of thymidine||–||+++