Chapter 15

Synchronization of Medicago sativa Cell Suspension Culture

Ferhan Ayaydin, Edit Kotogány, Edit Ábrahám,and Gábor V. Horváth

Abstract

Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques thatallow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell divisioncan be achieved using different plant tissues; however, well-established cell suspension cultures providethe largest amount of biological sample for further analysis. Here we describe the methodology of theestablishment, propagation, and analysis of a Medicago sativa suspension culture that can be used forefficient synchronization of the cell division and also the application and removal of hydroxyurea blockingagent. A novel method is used for the estimation of cell portion that enters S phase during the assay. Theprotocol can be used in the case of other species as well.

Key words: Medicago sativa suspension culture, cell cycle synchronization, hydroxyurea, 5-ethynyl-2′-deoxyuridine staining, fluorescence microscopy.

1. Introduction

Understanding the changes in the metabolic processes and in thestructure of plant cells during cell division requires a detailed anal-ysis of the cell cycle. This can be complicated because the cell cycleprogression in most somatic tissues is asynchronous and only aminor fraction of the cells are cycling. Therefore, different meth-ods have been developed for the enrichment of cells in a singlestage of the cell cycle to synchronize cells artificially. Differentplant tissues and cell cultures can be used to obtain synchronouslydividing cell populations.

Leaf mesophyll protoplasts from Petunia hybrida were shownto reenter the mitotic cell cycle from G1 phase after incubation in

G. Banfalvi (ed.), Cell Cycle Synchronization, Methods in Molecular Biology 761,DOI 10.1007/978-1-61779-182-6_15, © Springer Science+Business Media, LLC 2011

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a medium supplemented with growth regulators (1). This systemhas been used for other species (2, 3); however, its applicationmay be limited by the lack of protoplast isolation and cultureprocedures.

Root meristems can be a material of choice for a varietyof studies, since root tips are very rich in rapidly cycling cells.In Vicia faba more than 90% of the cells in the root meris-tems are cycling (4). Seedlings with actively growing roots canbe obtained from most plant species and it is easy to exposethe root tips to a great variety of nutritional, hormonal, andother chemical treatments. Reliable root meristem synchroniza-tion protocol is available (5) and this can be modified for differentspecies.

Rapidly growing suspension cultures are a suitable system tofollow cell cycle-associated changes in most biochemical or cyto-logical parameters because large number of cells are grown in anaqueous environment under well-defined conditions and they canbe exposed uniformly to synchronizing treatments. High degreeof synchrony could be achieved in cultured cells of Arabidopsis(6), carrot (7), tomato (8), and wheat (9). One of the most pop-ular systems is the tobacco BY-2 cell line which has an improved,efficient synchronization protocol (10).

Chemical methods of the synchronization of suspension cul-tures are reproducible and effective; these are based either on thedeprivation of an essential growth compound (6) or on the actionof chemical agents that blocks cell cycle progression. Hydrox-yurea is frequently used for root meristem and suspension culturesynchronization; this compound reversibly inhibits the ribonu-cleotide reductase enzyme and hence the production of deoxyri-bonucleotides (11). A treatment of suspension cells for 24–36 hat a concentration of 10–20 mM will lead to the accumulationof cells in G1 and early S phase. Aphidicolin, a fungal toxinderived from Cephalosporium aphidicola, is a specific inhibitorof the eukaryotic DNA polymerase and the plant α-like DNApolymerase arrests cycling cells also in G1/S phase, giving sim-ilar or slightly better results on suspension cultures than hydrox-yurea (7). Besides hydroxyurea and aphidicolin, a plant aminoacid mimosine can be used to block cell cycle traverse near theG1/S phase boundary by suppressing the formation of the rareamino acid hypusine in the eukaryotic translation factor 4D (12).This compound can be also efficiently used in plant suspensioncultures; in carrot cells it was found to be superior to aphidicolinor to hydroxyurea (7).

The detailed analysis of mitotic events may require a highlysynchronous population of the cells from M to the second Sthrough G1 phase. In such a case a two-step protocol can befollowed (10), in which the cells released from aphidicolin aresubsequently treated with propyzamide, a microtubule assembly

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inhibitor. However, the two-step protocol in this setup would notbe appropriate for analyzing metaphase-specific changes.

In the present protocol we give a detailed description forthe establishment of Medicago sativa cell suspension culture, thesynchronization procedure using hydroxyurea, and a novel invivo method for the determination of the fraction of cells enter-ing S phase using ethynyl-deoxyuridine (EdU)-based fluorescentstaining (13).

2. Materials

2.1. Cell Culturing 1. Murashige and Skoog Basal Salt Mixture (MS) – Plant tis-sue culture tested.

2. M. sativa cell culture medium: 4.3 g/l MS basal salt, 3%(w/v) sucrose, 100 mg/l myoinositol, 10 mg/l thiaminehydrochloride, 1 mg/l nicotinic acid, 1 mg/l pyridoxineHCl with 1 mg/l 2,4-D. Adjust the pH of the solution to5.8 with 1 M KOH and for plates the medium is solidifiedwith 0.8% Plant Agar.

3. Sucrose.4. Myoinositol: Prepare a 10 g/l stock solution (100× stock,

final concentration 100 mg/l) and store at 4◦C.5. Thiamine hydrochloride: Prepare a 100× stock solution at

1 g/l (final 10 mg/l) and store at 4◦C.6. Nicotinic acid (Free Acid): Prepare a 100× stock solution

at 100 mg/l (final 1 mg/l) and store at 4◦C.7. Pyridoxine HCl: Prepare a 100× stock solution at

100 mg/l (final 1 mg/l) and store at 4◦C.8. 2,4-Dichlorophenoxyacetic acid (2,4-D): Prepare 1,000×

stock solution (1 g/l, final 1 mg/l) and store at 4◦C.9. Kinetin (6-Furfuryladenine): Prepare 1,000× stock solu-

tion (0.2 g/l, final 0.2 mg/l), store at 4◦C.10. Plant Agar (Duchefa Biochemie).11. Sterile tissue culture equipment.12. Glass pipettes.13. Erlenmeyer flasks.14. Gyratory shaker and laminar flow hood.

2.2. Synchronization 1. Hydroxyurea: Prepare 1 M stock solution (final 10 mM,100× stock) and store at 4◦C (see Note 1).

2. Miracloth (or glass filter funnel with G3 pore size).

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2.3. Flow Cytometry 1. 5-Ethynyl-2′-deoxyuridine (EdU) stock solution: 10 mMEdU is prepared in dimethyl sulfoxide (DMSO) as a1,000× concentrated stock solution.

2. Nuclei isolation buffer: 45 mM MgCl2, 20 mM MOPS,30 mM sodium citrate, 0.1% Triton X-100, pH 7.0.

3. Sharp razor blades.4. Plastic dishes (6 cm in diameter).5. Nylon sieve (20 μm).6. Desktop centrifuge with swing-out rotor.7. Formaldehyde stock solution: Prepare 8% (w/v) stock solu-

tion by dissolving 8 g paraformaldehyde powder in 100 mlwater by heating to 60–70◦C in a fume hood (see Note 2).Add a drop of 5 M KOH to the solution to depolymerizeparaformaldehyde by heating at alkaline pH. After coolingto room temperature, adjust pH between 6.5 and 7.5 with5% (v/v) H2SO4. Aliquots of this stock solution can bestored at –20◦C for 6 months.

8. EdU detection cocktail: For each sample prepare 0.5 mldetection cocktail freshly (see Section 2.4).

9. Propidium iodide (PI) solution: Prepare 10 mg/ml PIstock solution in water (see Note 3).

10. FACSCalibur flow cytometer (Becton, Dickinson andCompany, NJ, USA) with the standard 488 nm laser fordetection of PI and AlexaFluor 488.

11. CellQuest software.

2.4. In Vivo EdUStaining, MitoticIndex Determination,and FluorescenceMicroscopy

1. Formaldehyde stock solution (8% w/v): See Section 2.3.2. Fixation solution (4% formaldehyde in PBS with Triton

X-100): Mix 8% formaldehyde stock solution with equalvolume of 2× PBS (2× PBS: 5.4 mM KCl, 2.94 mMNaH2PO4, 274 mM NaCl, and 16 mM Na2HPO4, pH 7.4)and add Triton X-100 to a final concentration of 0.1% (seeNote 4).

3. DNA staining (DAPI) solution: Prepare 1 mg/mlDAPI (4′,6-diamidino-2-phenylindole) solution in DMSO(10,000× stock) and dilute to 100 ng/ml in 1× PBS(2.7 mM KCl, 1.47 mM NaH2PO4, 137 mM NaCl, and8 mM Na2HPO4, pH 7.4) for staining nuclei (see Note 5).

4. EdU detection cocktail (Invitrogen, Click-iT EdU-AlexaFluor 488 HCS assay): For one sample reaction (167 μl),mix the following volumes of the kit components with144 μl distilled water: 1.6 μl buffer additive (Component F,kept frozen at –20◦C in small aliquots), 14 μl reaction buffer

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(Component D), 6.7 μl copper (II) sulfate solution (Com-ponent E, 100 mM CuSO4), and 0.07 μl Alexa Fluor 488azide (Component B, in 70 μl DMSO). Alternatively, a sub-stitute of the cocktail can be prepared by mixing the follow-ing components: 4 mM CuSO4, 40 mM sodium ascorbate,and 2.5–10 μM Alexa Fluor 488 azide (Invitrogen) in 1×PBS (for intact cells) or in nuclei isolation buffer (for nuclei).To prevent oxidation of the formed Cu (I) to non-catalyticCu (II) species, prepare the detection cocktail freshly anduse within an hour.

5. Mounting solution: For short-term mounting of samples use1× PBS which prevents cell shrinkage. For long-term preser-vation of samples use Fluoromount-G anti-fade mountingsolution (Southern Biotech).

6. Fluorescence microscope or confocal laser scanning fluores-cence microscope with appropriate filter sets.

3. Methods

3.1. Establishmentand Maintenance ofM. sativa CellSuspension Culturefor Synchronization

1. Initiate M. sativa ssp. varia (genotype A2) callus culturesfrom the hypocotyl explants of 7-day-old seedlings onM. sativa cell culture medium.

2. Subculture proliferative friable calli to 20 ml of the above-mentioned medium lacking Plant Agar and supplementedwith 0.2 mg/l kinetin.

3. After 2 and 4 weeks add 10–10 ml of fresh medium to theculture.

4. After this pour off 20 ml of the supernatant culture mediumand replace with 20 ml fresh medium weekly (no cells shouldbe wasted!).

5. Following four rounds of such subculturing increase the cul-ture volume stepwise: first dilute 25 ml of non-sedimentedculture to 50 ml with fresh culture medium in a 100 mlErlenmeyer flask.

6. Finally (after 3 months from the beginning of the suspensionculture) establish 100 and 200 ml cultures by weekly sub-culturing: dilute 50 ml non-sedimented culture to 100 ml(2× dilution) or 200 ml (4× dilution) by adding 50 or150 ml fresh culture medium in a 300 or 500 ml Erlenmeyerflask, respectively (see Note 6). Only well-established, rapidlydividing suspension culture can be used for a reproducible,efficient synchronization.

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7. Monitor the status of the suspension culture continuouslyas follows: pour 50 ml non-sedimented culture to a ster-ile graduated tube (for example, 50 ml CELLSTAR tubefrom Greiner Bio-One) and measure sedimented cell vol-ume. Indicate this value (12–15 ml for 4× diluted and18–22 ml for 2× diluted culture) on the flask; thus varia-tion in the sedimented volume of the culture (that shouldnot exceed ± 10%) can be recognized (see Note 6).

Different dilution ratios will result in suspension cultures of dis-tinct proliferative activity. Rapid cycling can be assayed by thedetection of the levels of cell cycle regulators in the proteinextracts of the cultures. The proteins examined can be cyclin-dependent kinases (particularly CDKB2;1-type plant-specifickinases that show G2/M phase-specific accumulation (14),cyclins, or other cell division regulators). Plant retinoblastoma-related proteins are also good choice for this purpose, since,for example, Arabidopsis thaliana RBR1 protein shows prolifer-ative activity-dependent accumulation in sucrose-starved suspen-sion cultures (15). We used the α-AtRBR1 C-terminal polyclonalantibody to detect changes in the level of MsRBR1 protein inalfalfa cell suspension cultures. Such approach can help to opti-mize not only the subculturing regime of the cell suspension butalso the correct timing of blocking agent treatment. G1/early Sphase blockers like hydroxyurea should be added to the culturethat is determined to undergo cell division but does not containhigh portion of cells in S phase, since hydroxyurea has an irre-versible toxic effect on these cells (16). According to our results,the 4× diluted culture should be blocked after 3 days of subcul-ture (see Note 6).

3.2. Synchronizationof M. sativa CellCulture withHydroxyureaTreatment

1. Add 2 ml 1 M hydroxyurea solution to 200 ml of an earlyexponential phase M. sativa ssp. varia (genotype A2) suspen-sion culture (4× dilution regime, 3 days after subculture)under sterile conditions. Efficient blocking of the cells needs36 h long treatment (26◦C, 130 r.p.m. shaking on a rotaryshaker in the dark).

2. Remove the blocking agent from the culture by excessivewashing. Use preconditioned medium for this purpose (seeNote 7). Filter the suspension culture on Miracloth placedin a glass funnel of appropriate size or on glass filter. Use onlygravitational flow of the culture medium and never allowthe suspension cells to dry completely. After the first filtra-tion, resuspend the cells gently but thoroughly (no clumpsshould remain in the suspension) in 200 ml preconditionedculture medium and filter them again. Repeat this washingstep two more times. Finally resuspend the cells in the orig-inal (200 ml) volume of the preconditioned medium.

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3. Take samples for flow cytometry (Section 3.3), in vivoEdU staining, mitotic index determination, fluorescencemicroscopy (Section 3.4), and RNA and protein analysis.For the last two purposes take 10 ml of the suspension,filter the cells on Miracloth, and dry them by pressing thecells wrapped in Miracloth gently to two–three layers of fil-ter paper. Divide the cells into microfuge tubes in 2:1 ratio;the bigger samples are for RNA isolation (give several hun-dred micrograms total RNA) and the smaller ones for pro-tein analysis (at least 100 μg protein can be isolated fromsuch samples).

A typical flow cytometric histogram of the cell cycle progres-sion of hydroxyurea-blocked alfalfa cell suspension is presentedin Fig. 15.1a. The data demonstrate that most of the cells enterG2/M phase between 12 and 14 h after the removal of theblocking agent and after 24–26 h the cells are ready to start anew round of cell division. A sharp increase in EdU-stained cellsfollowing hydroxyurea removal (Figs. 15.1b and 15.2) clearlyshows that the chemical block is reversible and the cells start DNAsynthesis simultaneously (see Note 8). The efficient staining andthe sharp change in the number of the fluorescent cells during cellcycle progression also demonstrate the usability of the presentedmethod for the calculation of cells entering S phase.

3.3. Analysis of CellCycle Parameterswith Flow Cytometry

1. Incubate 5 ml of alfalfa culture with 10 μM EdU for 1 h in6 cm Petri dishes by gentle rotation at 25◦C in the dark.

2. Centrifuge (5 min, 400×g) EdU-labeled and 0.1% DMSO-treated control cultures.

3. Chop the pellet with a sharp razor blade in 2 ml nuclei iso-lation buffer in 6 cm Petri dishes on ice.

4. Filter nuclei into 15 ml conical bottom tubes through 20 μmnylon sieves and fix on ice for 30 min by the addition of 8%formaldehyde solution to a final concentration of 1%.

5. Wash the fixed nuclei twice with 2 ml 1× PBS contain-ing 0.01% Triton X-100 (resuspension and centrifugation at4◦C, 10 min, 400×g) using a desktop centrifuge with swing-out rotor.

6. Incubate nuclei in 500 μl EdU detection cocktail for 30 minat room temperature in the dark.

7. Wash nuclei (4◦C, 10 min, 400×g) with 1× PBS containing0.01% Triton X-100 and counterstain either with 100 ng/ml(final concentration) DAPI (for microscopy check) or with5 μg/ml (final concentration) PI.

8. Analyze on a FACSCalibur flow cytometer with CellQuestsoftware. Use two fluorescence detectors with the stan-dard 488 nm laser. For Alexa Fluor 488-EdU intensity, use

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Fig. 15.1. Flow cytometric histogram analysis. (a) Cell cycle progression of cells syn-chronized at the G1/S boundary. AS: Asynchronous culture; BW: Before wash; AW: Afterwash. (b) Flow cytometric dual parameter analysis of the in vivo ethynyl-deoxyuridine-labeled cells (cells in S phase) synchronized at the G1/S boundary. Horizontal line at theleftmost panel indicates the EdU labeling threshold.

515–545 nm emission range (FL1 channel). For detectionof PI intensity (DNA content), use 564–606 nm emis-sion range (FL2 channel). Locate and gate nuclear pop-ulations by particle size using side scatter versus forwardscatter diagrams. To exclude particles which are not fluo-rescent with PI staining, use dot-plot diagram of “total PIfluorescence of a particle at FL2 channel” (FL2-A) versus“transit time of a particle at FL2 channel” (FL2-W) as sec-ondary gating. To locate the boundary of EdU-Alexa Fluor

Synchronization of Medicago sativa Cell Suspension Culture 235

Fig. 15.2. The EdU index (cells in S phase) and mitotic index of hydroxyurea-synchronized alfalfa cultures (see Note 3).

488-labeled nuclei in biparametric plots (EdU thresholdvalue), use counts versus FL1-H (Alexa488-EdU channel,log scale) histograms. The left (major) peak of this his-togram (with low green channel intensity) represents EdU-unlabeled G1 and G2 populations while the higher greenintensity second peak represents EdU-labeled nuclei. Todetermine the EdU threshold value, use the right borderof the leftmost major peak (where the unlabeled G1/G2counts reach zero value).

3.4. In Vivo EdUStaining, MitoticIndex Determination,and FluorescenceMicroscopy

1. Incubate 1 ml of alfalfa culture in a 2 ml microfuge tube ona roller with 10 μM final concentration of EdU for 1 h in itsown culture medium.

2. Settle cells by centrifugation (5 min, 400×g) and resuspendthe pellet in 2 ml 1× PBS and re-centrifuge. Discard thesupernatant.

3. Fix the cell pellet 15 min in 2 ml fixation solution on a roller.Replace the fixer with the same volume of 1× PBS by cen-trifugation. At this stage the cells can be kept in the refriger-ator for several weeks until further processing.

4. Following 3 × 5 min washes with 1× PBS (2 ml each), incu-bate 20–30 μl packed cell volume of cells in 167 μl EdUdetection cocktail by rotating for 30 min at room tempera-ture in a 0.2 ml microfuge tube (see Note 9).

5. After 2 × 5 min washes with 1× PBS (2 ml each) containing100 ng/ml (final concentration) DAPI, mount 20 μl aliquot

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Fig. 15.3. EdU-based replication assay on (a) asynchronous and (b) hydroxyurea-synchronized (after wash sample) alfalfa cells. Transmission images (grayscale) areoverlaid onto AlexaFluor488-labeled (pseudocolored white) nuclei. Bar 10 μm.

of cells onto microscope slide with a coverslip and gentlypress with a tissue paper to flatten the clusters. The rest ofthe labeled cells can be kept at 0–4◦C in a dark container forseveral weeks (see Note 10).

6. Set the confocal microscope configuration as follows: objec-tive lenses: UPLSAPO 20× (dry, NA 0.75) and UPLFLN40× (oil, NA 1.3); sampling speed: 4 μs/pixel; line aver-aging: 2×; scanning mode: sequential unidirectional; excita-tion: 405 nm (DAPI) and 488 nm (Alexa Fluor 488); lasertransmissivity: less than 1% for DAPI and 5% for Alexa Fluor488, respectively; main dichroic beamsplitter: DM405/488;intermediate dichroic beamsplitter: SDM 490; DAPI fluo-rescence detection: 425–475 nm; Alexa Fluor 488 detection:500–600 nm. For transmitted light imaging (Fig. 15.3)differential interference contrast (DIC) mode can be used.Count more than 500 cells for mitotic index and EdU label-ing index determinations.

4. Notes

1. Caution: Hydroxyurea is toxic and carcinogenic; use appro-priate precautions.

2. Paraformaldehyde is very hazardous in case of skin contact,eye contact, or inhalation (irritant/corrosive). Work in afume hood and wear protective equipment.

3. Caution: Propidium iodide is a known mutagen; use appro-priate precautions.

4. The addition of Triton X-100 to the fixation solution pro-vides uniform fixation with reduced cell shrinkage.

Synchronization of Medicago sativa Cell Suspension Culture 237

5. Caution: DAPI is a known mutagen; use appropriateprecautions.

6. Careful propagation of the cell culture is an absolute pre-requisite of efficient synchronization. Since the establish-ment of a culture may exceed the described time demandthis should be started well before the planned synchro-nization. For scientific purposes established cultures ofM. sativa ssp. varia (genotype A2) can be obtained fromDr. Gábor V. Horváth (Institute of Plant Biology, Bio-logical Research Center HAS, Szeged, Hungary, e-mail:hvg@brc.hu).

7. Use of preconditioned medium in the washing steps andfor the final resuspension will greatly increase the successof synchronization. To obtain this, the same M. sativa sus-pension culture should be grown under the same subcul-ture regime, and just prior to the beginning of hydroxyurearemoval, four times 200 ml of this culture should be filteredthrough sterile glass filter. Cells can be collected and used asunblocked control; the resulting preconditioned medium isused to remove hydroxyurea from blocked suspension.

8. Relatively high labeling efficiency can be detected in“Before Wash” samples, although the labeling intensity isvery low compared to the other samples. Since EdU is athymidine analog, cells with depleted nucleotide pools (dueto the action of hydroxyurea) start to incorporate EdU;however, the DNA synthesis cannot proceed due to thelack of other nucleotides.

9. Fluorochrome-containing solutions should not be exposedto strong light; therefore, labeling reaction should be per-formed at dark or under dim light. The simplest solution isto wrap the centrifuge tubes in aluminum foil for the timeof the labeling.

10. Glycerol-based (or high osmolarity) mounting media maycause cell shrinkage but they better suit imaging with oilimmersion objectives. Mounting the samples in PBS pre-vents cell shrinkage; however, care must be taken not todry out the sample. Occasional PBS loading may be neces-sary for prolonged observations to prevent sample drying.

Acknowledgment

The authors are grateful to Katalin Török for excellent technicalassistance. This work was funded by OTKA grant no. NK 69227.Edit Ábrahám was supported by the János Bolyai Fellowship ofthe Hungarian Academy of Sciences.

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