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Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2′-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments. © 2008 International Society for Advancement of Cytometry
FCM is one of the most commonly used techniques for studying protein expression at the level of single cells, in particular where multiparametric (1, 2) and multicolor (3) analyses are used as a readout. Integration of FCM into proteomics technologies have played a key role for cell functions and proliferation studies using “cytomics” approches (4, 5). Incorporation of the thymidine analogue BrdU into DNA of proliferating cells is widely used to assess the cell cycle status of cells using different methods, such as image cytometry, HCA, and FCM (6, 7).
A downside of this technique is that detection of BrdU requires DNA denaturation causing many epitopes to become modified or destroyed (8–12), resulting in classical antibody staining methods for multiplex analysis no longer possible (11, 12).
To address this issue we have setup a novel method for identifying S-phase cells that overcomes the DNA denaturation step but still detects BrdU.
The “Click Chemistry” bio-conjugation reaction using the Sharpless cycloaddiction of azides and acetylenes catalyzed by Copper (I) (CuAAC) is widely used in drug discovery, for example for protein tagging (13) or DNA sequencing (14). The method has favorable thermodynamic properties accompanied by high specificity and a high quantitative yield of the end product in aqueous solvents at physiological temperatures, providing 1,4-disubstituited triazoles (15).
In our method, using CuAAC, we were able to detect alkynyl tags from incorporated 5-Ethynyl-2′-deoxyuridine (EdU) during DNA synthesis by reactive BrdU azides under conditions preserving protein epitopes. The resulting bromouracil moiety can then be detected by anti-BrdU mAb. Here we describe the assay development and validation for FCM and image cytometry, including HCA. The combination of the click chemistry reaction with good mAb specificity and an amplification step by AlexaFluor® secondary step are the key points for the high specificity and sensitivity of this new approach.
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Since the introduction of the BrdU technique in 1982 by Gratzner, BrdU immunostaining procedures have required denaturation of chromatin to enable mAb binding to incorporated BrdU (6, 7). Usually, the denaturation step is performed by pretreating samples with chemicals, but such harsh procedures can alter cell morphology and interfere with the simultaneous immunostaining of other cellular antigens (11, 12). Other approaches used to solve this issue, such as enzymatic treatment by nucleases or UV-B photolysis, have disadvantages in terms of cost, reproducibility, and sensitivity (8–10).
In this article we have shown an interesting alternative approach for proliferation assays without a denaturation step. In our method, we coupled the terminal ethynyl tag (19, 20) of incorporated EdU to BrdU azide derivatives using a click chemistry reaction, followed by detection using an anti-BrdU mAb and fluorescence amplification in the secondary Ab staining step. Since our azide probes are still not commercially available, we also describe a rapid and convenient chemical protocol for producing BMA/BDA azides from any BrdU source. (see Online Supplementary Materials).
EdU showed a slightly increased but mild anti-proliferative activity compared to BrdU at continuous treatments (72 h) in a classical anti-proliferation assay. However, when cells were pulsed for 1 h and kept in drug-free medium in the same conditions, growth inhibition was negligible.
Previous studies (21–23) have shown that EdU increases growth of thymidine synthetase deficient cells as it acts as a thymidine analog, but conversely antiviral activity similar to other pyrimidine nucleoside analogs was observed due to its activity against deoxynucleoside kinases and thymidine synthetase. EdU modified primers showed an increase of Tm in RT-PCR reactions and an increased stabilization of the DNA duplex (24). This could explain a postreplicative toxicity after EdU incorporation during DNA synthesis. However, the precise mechanism of action of these modified nucleotides is still not well documented. Since our method is very sensitive with respect to the detection of ethynyl residues, very low concentrations of EdU (1 μM) used for a short pulse (20 min) were sufficient for the readout and reduce the risk of side effects. The BrdU azide reaction was found to be very quick and saturation was reached in less than 10–15 min. One of the major advantages of the method is that fluorescence from AlexaFluor® 488 linked antibodies after BMA/BDA ligation was minimally quenched by the commonly used DNA dye PI. On the contrary, fluorescence emission from direct ligation of AlexaFluor® 488 Click-IT™ was significantly quenched, making FCM applications in combination with PI more difficult. The greater molecular distance between the PI intercalated into DNA and the AlexaFluor® 488 fluorochrome linked to the secondary Ab in the indirect staining method might be the reason for the reduced quenching. This could decrease the risk of energy-transfer due to the spectral behaviors of the fluorescent dyes. Another advantage is that our method raises the possibility of using a broad spectrum of conjugated secondary antibodies for multicolor analysis (3, 4), which overcomes the limitation of fluorescence dye azide availability. This could also be advantageous when using HCA methods such as ArrayScan Cellomics readers, which are highly sensitive and robust when used with our method.
Different behaviors were seen for BMA and BDA, with a brighter signal observed for BDA. This might be due to improved chemical characteristics. In fact, it was shown that the 1,3-bis (azido) chemical class is more reactive than the corresponding azide (25). This could result in an increased activity against ethynyl tags in less accessible or more compact chromatin producing more alkynyl tags.
Since the major advantage of this new method, when compared with BrdU incorporation, is the option for performing multiplex analysis by click coupling of BMA or BDA, we analyzed HL-60, A2780 and U2-OS cells simultaneously for cell cycle progression and phosphorylation of histone H3 on Ser10 or induction of apoptosis by following cleavage of caspase-3 in the presence of different cytotoxic drugs. We showed differential staining patterns of these cell lines, which are in line with the expected phenotypes.
Finally, our method is also applicable for in vivo applications. We have shown that EdU administration in vivo can be used for FCM studies in isolated bone marrow cells by staining different intracellular epitopes with high sensitivity. EdU is very well tolerated and even at high doses (tested up to 50 mg/kg) no signs of toxicity were seen. The possibility of using low concentrations of EdU (down to 5 mg/kg) for detection of the signal assures a robust window for tolerability in vivo, much wider than for BrdU.
Finally, the major properties of the different methods in comparison to the classical BrdU assay are summarized in Table 1. The click chemistry methods using EdU do not require DNA denaturation since the chemical reaction was performed at physiological conditions and was compatible with multiple mAb staining and multicolor analysis including HCA applications. In addition BrdU coupling by CuAAC was faster, exhibited a higher signal to noise ratio and was less affected by PI quenching than direct ligation by fluorochrome azide.
Table 1. Summary table for comparison of BrdU classical assay and Click Chemistry approches based on direct ligation using Click-IT™ technology (AlexaFluor® 488) and indirect coupling by BrdU azides
| ||BrdU INDIRECT||EdU + CLICK-IT™ ALEXA 488 “DIRECT”||EdU + BrdU AZIDE “INDIRECT”|
|Antiproliferative activity in vitro||Negligible||Weak (1)||Weak (1)|
|DNA incorporation in vivo (BM)||Yes||Yes||Yes|
|Assay time setup||+++||+||++|
|CuAAc reaction time||N/A||>30 min||Short|
|Quenching after PI staining||+/−||++||+/−|
|Multiplex analysis by MAb||+/−(2)||Yes||Yes|
|Polychromatic analysis||++||+ (3)||++|
|Signal to noise ratio (sensitivity)||++||++||++/+++ (4)|
|Suitable for imaging/HCA||−||+||++|
Our approach based on BrdU coupling by click chemistry ligation followed by mAb detection could potentially be interesting not only for cell cycle studies but also for a number of additional readouts. Putative applications envisaged are the use of ethynyl modified oligonucleotide (14, 24) for siRNA interference studies, localization of engineered proteins bearing pEthynyl-Phe aminoacids (13) or for detection of alkynyl chemical tags in modified substrates (20, 26) expanding FCM and image cytometry analytical capability (27–29).