Abstract
BackgroundProgress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.ResultsApplications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice.ConclusionsIn plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.
Highlights
Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA
Since EdU detection does not rely on antibodies and the molecular size of the chemicals used during detection is small compared to the size of the antibody molecules, cells were directly incubated with the detection cocktail without performing cell wall digestion
Omission of cell wall digestion and DNA denaturation/digestion steps resulted in better preservation of cellular structures judged by DAPI (4’,6-diamidino-2-phenylindole) staining of the nuclei and by differential interference contrast (DIC) microscopy (Figure 1A)
Summary
The most accurate method utilizes direct measurement of new DNA synthesis This has been performed by incorporating tritium-labeled thymidine and detection by autoradiography [1]. Because of the involvement of radioactivity, this method has been replaced by incorporation of a thymidine analog such as bromodeoxyuridine (BrdU) into DNA, followed by immunodetection with a specific antibody raised against the thymidine analog [2]. Being effective, this method requires DNA denaturation or digestion To alleviate the stress-related artifacts, it is possible to first chemically fix the cells and partially digest cell walls. This approach requires highly pure and expensive cell wall digestion enzymes, as crude enzyme preparations contain impurities such as proteases and nucleases that can significantly compromise cellular integrity [5]
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