A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine
© Kotogány et al; licensee BioMed Central Ltd. 2010
Received: 5 August 2009
Accepted: 28 January 2010
Published: 28 January 2010
Progress 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.
Applications 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.
In 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.
Detection of cell proliferation is a fundamental method for assessing cell health, determining genotoxicity, and evaluating stress responses. The most accurate method utilizes direct measurement of new DNA synthesis. Traditionally, this has been performed by incorporating tritium-labeled thymidine and detection by autoradiography . Because of the involvement of radioactivity, this method has been replaced by incorporation of a thymidine analog such as bromodeoxyuridine (BrdU) into DNA, followed by immunodetection with a specific antibody raised against the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 . Although being effective, this method requires DNA denaturation or digestion (using hydrochloric acid, heat or DNase) to expose BrdU to the antibody. This step is lengthy, difficult to perform consistently, and can adversely affect the morphology of the sample. Antibody-based detection method of BrdU assay also necessitates cell wall digestion in experiments carried out on plant cells. Therefore protoplasts, partially cell-wall-digested cells and organs or tissue sections are often used for BrdU-based detection of proliferative activity in plants .
However, treatment with cell wall digesting enzymes imposes a significant wounding and osmotic stress on plant cells. Moreover, types and concentrations of the enzymes and the osmolarity of the digestion medium should also be specifically optimized for each plant species, organ and cell type under investigation . Partial cell wall digestion or release of protoplasts not only prolong the experimental duration but also cause substantial reorganization of cytoskeleton and activation of stress and defense-related genes. To alleviate the stress-related artifacts, it is also possible to first chemically fix the cells and then partially digest cell walls. However, 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 .
EdU (5-ethynyl-2'-deoxyuridine) is a terminal alkyne-containing nucleoside analog of thymidine, and is incorporated into DNA during active DNA synthesis . EdU detection method is based on click chemistry . In a Cu(I)-catalyzed reaction, the alkyne of EdU reacts with an azide containing fluorochrome, forming a stable covalent bond. EdU-based assay has been successfully used in detection of proliferation in avian cochlea , in chick embryos  in breast cancer cells  and in human fibroblasts . Twenty-four hours long EdU incubation duration has been recently used in Arabidopsis root tips to identify dysfunction of the quiescent center , but the possibility of very short EdU pulse labeling to determine S-phase indices, the suitability of this novel detection method in various plant species, comparison of EdU assay to BrdU assay and plant specific parameters and fields of application such as plant flow cytometry have not been explored in detail.
Here we show the advantages in microscopy and flow cytometry applications of this novel S-phase detection assay using various cultured plant cells and root meristems.
Results and Discussion
EdU-based assay versus immunodetection of BrdU
Comparison of experimental steps and durations for EdU-assay and BrdU-based immunolocalization
EdU or BrdU labeling
Fixation and washes
15 min + 15 min
15 min + 15 min
Cell wall digestion and washes
30 min + 15 min
Plasma membrane permeabilization and washes
30 min + 15 min
DNase I digestion and washes
30 min + 15 min
EdU click reaction or BrdU antibody incubations
3 h + 25 min + 1 h
Washing with DAPI and sample mounting
Total experimental duration
3 h 20 min
9 h 30 min
EdU assay is a versatile method for both monocot and dicot plant species
Using EdU assay in flow cytometry of plant nuclei
Based on these experiments, the advantages of EdU assay for use in plant flow cytometry were readily evident. Clearly defined EdU-labeled population on a logarithmic scale in biparametric plots allowed us to precisely determine the fraction of cells labeled with EdU, hence giving information on the dynamics of S-phase entry and progression of cells into G2 phase of the cell cycle. Especially with short EdU pulse durations, dual parameter analysis with EdU is very informative and advantageous as subpopulations of S-phase (early, mid and late S-phase) can be better assessed. As an example, Figure 5B displays a simple quadrant analysis. Q1 and Q2 sectors indicate the population of 2C and 4C nuclei that never entered into replication phase during EdU labeling period. Q3 fraction represents nuclei entered into S-phase relatively recently. Q4 fraction indicates nuclei entered into S-phase earlier than that of Q3 fraction during the EdU labeling period. This fraction contains nuclei in mid to late S-phase in addition to EdU-labeled nuclei that already entered into G2 phase with 4C DNA content.
In summary, the small size of molecules participating in the EdU labeling and detection allows for fast and efficient detection of the incorporated EdU without using harsh conditions, which may adversely affect the quality of data especially in plant flow cytometry. Simple and efficient EdU-based biparametric flow cytometry can therefore be readily incorporated into studies involving both monocot and dicot plant species and can be a very effective tool to assess culture health and proliferation status or in experiments based on plant cell cycle synchronization, stress treatments and genetic modifications.
EdU analysis of S-phase cells in rice root meristems
The EdU-based S-phase assay presented here affords a simple and rapid yet robust complement to previously validated methods of proliferation analysis in plant cells. Unlike antibody-based BrdU assay, EdU-based S-phase assay does not require cell wall digestion or DNA denaturation. This is not only advantageous from the point of morphological preservation of the samples, but also saves considerable amount of experimental time. EdU labeling and detection protocol can easily be adapted to various plant species regardless of cell wall thickness, composition or structure. The assay will be particularly useful in plant flow cytometry analyses due to superior preservation of isolated nuclei during the detection protocol. Apart from its application in cultured cells and isolated nuclei, the method is also well suited for quantitative proliferation analysis on thick tissues such as roots. Taking these data into account, we conclude that practical, versatile and quantitative nature of this robust S-phase assay will find its application in various fields of plant science research and will certainly be the new gold standard replacing BrdU-based immunolocalization assays.
Plant growth conditions
Hormone concentrations and media used for plant cultures.
Alfalfa (M. sativa ssp. Varia A2)
Arabidopsis (A. thaliana ecotype Columbia)
Grape (V. berlandieri × V. rupestris cv. 'Richter 110')
Maize (Z. mays, cv. H1233)
N6 M (LP40)
Rice (O. sativa ssp. japonica cv. 'Unggi 9'
Tobacco (N. tabacum cv. Petit Havana SR1)
Immunolocalization of BrdU
BrdU (5-bromo-2'-deoxyuridine, Sigma catalog no: B5002) stock solution was prepared as 30 mM aliquot in DMSO and kept in freezer. Two-days-old suspension culture of Arabidopsis was incubated for 2 hrs with 10 μM BrdU in its own culture medium. Cells were then fixed 15 min in 4% (w/v) formaldehyde solution in phosphate buffered saline (PBS) with 0.1% Triton X-100. Eight percent (2× concentrated) formaldehyde stock solution was prepared as following: Paraformaldehyde powder (Fluka cat. no: 76240) was dissolved in water by heating to about 60°C inside a fume hood and a drop of concentrated KOH was added as heating and alkaline pH depolymerizes paraformaldeyde. After cooling to room temperature, pH was set to neutral pH with dilute H2SO4 . Aliquots of this stock fixer can be frozen for a few months. This 2× formaldehyde solution was then mixed 1:1 with 2× PBS (1× PBS contains 2.7 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8 mM Na2HPO4, pH7.4) and Triton X-100 was then added to a final concentration of 0.1% which provides uniform fixation with reduced cell shrinkage. Fixed cells were washed 2 × 5 min with PBS and 1 × 5 min with 0.5% MES (2-N-morpholinoethanesulfonic acid) pH 5.8. Cell walls were partially digested 30 min with chromatographically purified lyophilized enzymes from Worthington Biochemical Corporation (Lakewood, NJ, USA). The enzyme mixture was 1% cellulase (Cat no: LS02601) and 0.5% pectinase (Cat. no: LS04297) in 0.5% MES, pH 5.8. After washing with PBS (3 × 5 min), cells were settled on poly-L-lysine coated multiwell slides, excess solution was removed and cells were permeabilized 30 min with 0.5% Triton X-100 in PBS to allow antibody penetration. Following 3 × 5 min washes with PBS containing 5 mM MgSO4, cells were incubated 30 min in 20 units/ml chromatographically purified, ribonuclease- and protease-free DNase I (Worthington Biochem. Corp. cat. no: LS006331) in PBS with 5 mM MgSO4. Fifty times concentrated stock solution of DNase I (1000 units/ml) was prepared by dissolving 2500 units of lyophilized powder in 1.25 ml 50% glycerol with 1 mM CaCl2 and kept in freezer in aliquots. DNase I solution was removed with 3 × 5 min washes with antibody buffer. Antibody buffer, (PBS+) contains PBS with 5% (v/v) fish gelatin (Sigma cat. no: G7765, to prevent nonspecific binding of antibodies) and 0.02% (w/v) sodium azide (Fluka cat. no: 71290, to inhibit bacterial growth during antibody incubations. For EdU assay sodium azide should not be used before click reaction). Cells were incubated 3 h at 37°C with monoclonal (clone BU-33) mouse anti-bromodeoxyuridine antibody (Sigma cat. no: B8434) diluted 1:200 in PBS+. Following 5 × 5 min washes with PBS+, cells were incubated 1 h at 37°C with rabbit anti-mouse Alexa Fluor 488 conjugated antibody (Invitrogen cat no: A11059) diluted 1:300 in PBS+. Cells were then washed 3 × 5 min with PBS containing 100 ng/ml DNA staining dye DAPI (4',6-diamidino-2-phenylindole, Invitrogen cat no: D1306) and mounted with Fluoromount-G anti-fade mounting solution (Southern Biotech, cat no: 0100-01).
EdU-based proliferation assay
Two-days-old monocot and dicot plant suspension cultures were incubated 2 hrs with 10 μM EdU (Invitrogen cat no: A10044) in their own culture medium. Arabidopsis cultures (36 h-old) were incubated with various concentrations and durations as shown in Figure 4. Cells were then fixed 15 min in 4% (w/v) formaldehyde solution in phosphate buffered saline (PBS) with 0.1% Triton X-100. Addition of the detergent Triton X-100 in the fixer prevents cell shrinkage and it also partially permeabilizes the plasma membranes for small detection reagents of EdU assay. Moreover, quick penetration of the fixer allows better preservation of mitotic chromosomes. However, for experiments where preservation of cytoskeleton or cytoplasmic organelles is important, detergent should be omitted from the fixer and plasma membranes should be permeabilized after fixation as in BrdU immunolocalization protocol. Following 3 × 5 min PBS washes, 20-30 μl packed cell volume of cells were directly incubated 30 min at room temperature (RT) in EdU detection cocktail (Invitrogen, Click-iT EdU Alexa Fluor 488 HCS assay, cat no: A10027). For 1 sample reaction, following amounts of the kit components are mixed in 144 μl distilled water: 1.6 μl buffer additive (component F, kept frozen in small aliquots), 14 μl reaction buffer (Component D), 6.7 μl Copper (II) sulfate solution (Component E, 100 mM CuSO4) and 0.07 μl Alexa Fluor 488 azide (Component B, in 70 μl DMSO). Many azide-labeled fluorochromes other than Alexa Fluor 488 are available throughout the visible spectrum for multicolor labeling purposes. Click reaction requires Cu (I) which can be formed using CuSO4 in the presence of a reducing agent such as sodium ascorbate . For experiments where defined buffer components are necessary, we have found that the use of detection cocktail with the following composition resulted in positive EdU labeling on Arabidopsis suspension cultures and on isolated rice and alfalfa nuclei: 4 mM CuSO4, 40 mM sodium ascorbate, 20 μM Alexa Fluor 488 azide in PBS (for intact cells) or in nuclei isolation buffer (for nuclei). To prevent oxidation of Cu (I) to non-catalytic Cu (II) species, the detection cocktail should be prepared freshly. Although the click reaction is not light sensitive, fluorochrome-containing solutions should not be exposed to strong light. After 2 × 5 min washes with PBS containing 100 ng/ml DAPI, an aliquot of cells were mounted in PBS. Glycerol-based (or high osmolarity) mounting mediums caused shrinkage during mounting; therefore PBS mounting is used for all cell lines. For root tip labeling, root tips of germinating rice seedlings (O. sativa L. ssp. japonica 'Nipponbare') were submerged into 20 μM (Figure 6) or 0, 2, 20, 100 μM EdU (Figure 7) in half strength MS medium. Root tips were then cut in detergent-containing fixer (see above) and fixed for 30 min at RT. Fixer was washed with PBS (3 × 10 min) and root tips were incubated in EdU detection cocktail (see above) for 30 min followed by PBS or PBS-DAPI washes (see above). Fluoromount-G anti-fade solution was used for mounting of root tips.
Confocal laser scanning and fluorescence stereo microscopy
Confocal laser scanning microscopy was performed using Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus Life Science Europa GmbH, Hamburg, Germany). Microscope configuration was the following: objective lenses: UPLSAPO 20× (dry, NA:0.75), UPLFLN 40× (oil, NA:1.3) and UPLSAPO 60× (oil, NA:1.35); sampling speed: 4 μs/pixel; line averaging: 2x; scanning mode: sequential unidirectional; excitation: 405 nm (DAPI and cell wall lignin autofluorescence) and 488 nm (Alexa Fluor 488); laser transmissivity: less than 1% and 5% were used for DAPI and Alexa Fluor 488, respectively; main dichroic beamsplitter: DM405/488; intermediate dichroic beamsplitter: SDM 490; DAPI and cell wall autofluorescence were detected between 425-475 nm and Alexa Fluor 488 was detected between 500-600 nm and pseudocolored red and green, respectively. Differential interference contrast (DIC) images were captured with 488 nm laser line. For rice root tip imaging and EdU-signal quantitation, single optical sections of 6 μm (optical depth) on the median plane of rice root tips were captured with 20× objective. Identical laser power and detection settings were used for quantitative analyses. For imaging of all root tips of Figure 7, a higher detector sensitivity setting (as compared to Figure 6) was used due to very short EdU pulse durations. The center of the root with the widest girth was determined by lignin autofluorescence signal of the cell walls. For quantitation of the green EdU signal, meristem regions were manually traced up to 500 μm distance starting from the quiescent center. Area measure tool of Olympus Fluoview software (version 184.108.40.206) was used for measurement of area and intensities at the root tips. Total intensity of the green signal (named as "integration" by Olympus Fluoview software) was divided by the measured meristem area to determine average intensity per square microns and plotted using Microsoft Office Excel 2003 software. For nuclear counterstaining and close-up view of chromosomes, DAPI (200 ng/ml) was used on EdU labeled (2 hrs) roots and observed by 40× objective. For fluorescence stereo microscopy, Olympus SZX12 stereo microscope was used. Alexa Fluor 488 images were captured with the "green" filter set: Excitation: 460-490 nm, dichroic beam splitter 505 nm, emission: 510-550 nm. Transmission photos were pseudocolored red and merged with fluorescence images. Composite images were prepared using "import image sequence" and "make montage" functions of ImageJ software (National Institutes of Health, USA, version 1.41).
Nuclei isolation and Flow cytometry
Three-days-old rice and alfalfa cultures were incubated with 10 μM EdU for 8 hrs and 4 hrs, respectively. For unfixed nuclei preparations, 4d-old rice cells were 10 μM EdU-treated for 4 hrs. Three-days-old Arabidopsis cultures were incubated either with 0.1% DMSO (as control) or with 10 μM EdU in DMSO for 15' and 30'. EdU-labeled and 0.1% DMSO-treated control cultures were chopped with a sharp razor blade in nuclei isolation buffer (45 mM MgCl2, 20 mM MOPS, 30 mM sodium citrate, 0.1% Triton X-100, pH 7.0) in 6 cm plastic Petri dishes on ice . Nuclei (in 2 ml buffer) were filtered into 15 ml conical bottom tubes through 20 μm sieves and fixed on ice for 30 min by the addition of 8% formaldehyde solution (see above) to a final concentration of 1%. Fixed nuclei were washed twice with 2 ml 0.01% Triton X-100 containing PBS by centrifugation at 4°C (10 min, 400 g/1500 rpm) using Heraeus Labofuge 400R (Thermo Fisher Scientific, Rockford, IL, USA) desktop centrifuge with swing-out rotor. Washed nuclei were incubated in 500 μl EdU-detection cocktail for 30 min at room temperature. Unfixed nuclei were centrifuged (10') and resuspended in 500 μl EdU-detection cocktail and incubated 30' at room temperature (Water and buffer component D in the cocktail recipe were replaced by nuclei isolation buffer for unfixed nuclei). After one wash (5') with PBS containing 0.01% Triton X-100 (for fixed nuclei) or with nuclei isolation buffer (for unfixed nuclei), nuclei were counterstained either with 100 ng/ml DAPI (for microscopy check) or with 5 μg/ml PI (propidium iodide, Invitrogen cat. no: P1304 MP) and analyzed on a FACSCalibur flow cytometer (Becton, Dickinson and Company, NJ, USA) with CellQuest software. Two fluorescence detectors are used with the standard 488 nm laser. Alexa Fluor 488-EdU intensity was detected between 515-545 nm (FL1 channel). For detection of PI intensity (DNA content) 564-606 nm emission range was used at FL2 channel. Side scatter versus forward scatter diagrams were used to locate and gate nuclear populations by particle size. Dot-plot diagram of "total PI fluorescence of a particle at FL2 channel" (FL2-A) versus "transit time of a particle at FL2 channel" (FL2-W) was used as secondary gating to exclude particles which are not fluorescent with PI staining. To locate the boundary of EdU-Alexa Fluor 488-labeled nuclei in biparametric plots (EdU threshold value) counts versus FL1-H (Alexa488-EdU channel, log scale) histograms were used. The left (major) peak of this histogram (with low green channel intensity) represents EdU unlabeled G1 and G2 populations while the higher green intensity second peak represents EdU-labeled nuclei. The right border of the leftmost major peak (where the unlabeled G1/G2 counts reach zero value) is selected as the EdU threshold value. EdU threshold values of control samples were determined by corresponding EdU-treated samples. The midpoint PI intensity (~300 PI intensity units) is selected as the vertical separating line for 2C DNA and 4C DNA contents in quadrant analyses.
We would like to thank Katalin Török for technical assistance with plant cell cultures and Zsuzsanna Kószó for technical assistance during experiments involving microscopy. This work was funded by the Hungarian National Research Foundation (OTKA grant no: NK69227)
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