chapter 3 · concentrations of nonionic detergents such as triton x-100 . ... less steel grinder...

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Nicolas L. Taylor and A. Harvey Millar (eds.), Isolation of Plant Organelles and Structures: Methods and Protocols, Methods in Molecular Biology, vol. 1511, DOI 10.1007/978-1-4939-6533-5_3, © Springer Science+Business Media New York 2017

Chapter 3

Isolation of Nuclei and Nucleoli

Alison F. Pendle and Peter J. Shaw

Abstract

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purifi ed for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Key words Nuclei , Nucleoli , Nuclear isolation , Immunofl uorescence , Proteomics

1 Introduction

Nuclei are usually the most easily identifi able subcellular organelles in plant cells. They vary greatly in size depending on the genome size of the species and the degree of ploidy of the cell type. Meristematic nuclei are generally fairly round, whereas nuclei from differentiated cells can be quite elongated. Nuclei have a large refrac-tive index because of their high chromatin content, and this makes them easily visible by phase contrast or differential interference con-trast microscopy . Their high DNA content means that they are brightly stained by DNA-binding dyes. The fl uorescent dye DAPI (4′,6-diamidino-2-phenyl-indole) is a particularly useful dye for visualizing nuclei, since its fl uorescence increases greatly when bound to DNA; this means that DNA can be visualized with a very low background. DAPI’s excitation maximum is at 358 nm, although for microscopy longer near UV wavelengths are used as most micro-scope objectives do not transmit such short wavelengths. The fl uo-rescence emission is an intense blue with a maximum at 461 nm.

Any preparation of nuclei or subnuclear structures should be closely monitored at all stages by microscopy; a simple phase con-trast microscope will be suffi cient if a fl uorescence microscope is not easily available. Figure 1 shows nuclei from different plant spe-cies imaged by phase contrast and fl uorescence microscopy using DAPI. The nuclei are characterized by bright DAPI staining, with

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one or more dark areas that correspond to the nucleoli, which are also clearly visible in phase or differential interference contrast because of their different refractive index. Other subcellular organ-elles such as chloroplasts and mitochondria are common contami-nants of nuclear preparations, but the most common contaminants are starch granules. These are very dense, and thus tend to copurify with nuclei at the bottom of density gradients, or form a pellet at the bottom of a centrifuge tube, often puncturing the nuclei on their way down. They can be distinguished from nuclei by their lack of labeling with DAPI.

The detailed protocol for nuclear and nucleolar preparation depends on the purpose for which the nuclei are required. The pro-tocols described here have been mainly used for biochemical studies, such as proteomic analysis [ 1 ]. The fi rst stage is to produce a suspen-sion of the nuclei, which necessarily involves breaking open the cells in some way. Plant cells are enclosed in a rigid cell wall , which must be physically opened or broken by mechanical disruption, or alterna-tively removed by treatment with cell wall degrading enzymes to produce protoplasts . Mechanical disruption is generally needed to release the nuclei from protoplasts. We have usually used a stainless steel homogenizer with a carefully engineered clearance ( see Note 1 ). The suspension of nuclei is then purifi ed for biochemical studies,

Fig. 1 Nuclei from different plant species imaged by phase contrast microscopy ( a , c , e ) and DAPI staining and fl uorescence microscopy ( b , d , f ). ( a , b ) Arabidopsis thaliana root nuclei. ( c , d ) Wheat ( Triticum aestivum ) root nuclei. ( e , f ) Pea ( Pisum sativum ) root nuclei. Bar = 20 μm


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although it can be used directly for microscopy , after spinning down on to a slide or coverglass using a cytospin centrifuge [ 2 , 3 ]. Apart from starch granules, nuclei are the densest subcellular organelles and are often purifi ed by centrifugation in a medium or gradient of the right density. Isolated nuclei are very fragile and easily broken open; they need to be handled very gently, preferably centrifuging on to a cushion of higher density to avoid being damaged by hitting the bottom of the centrifuge tube. Nucleoli can be obtained by con-tinuing homogenization of the nuclei (Fig. 2 ). Again, the progress of homogenization should be frequently monitored by phase microscopy of samples of the suspension.

The method for preparation of nuclei given here is broadly based on that of Saxena et al. [ 4 ], particularly the use of lower pH buffers than used for animal nuclei, which improves the stability of plant nuclei. DNA is generally complexed with Mg 2+ ions, and the inevitable breakage of nuclei during isolation causes some of this to spill out and become cross-linked into an unworkable mass from which it is impossible to purify nuclei. Cook and colleagues [ 5 ] suggest encapsulating cells in agarose beads to prevent this, before dissolving away the cytoplasm. While this is a reasonable strategy for some purposes, it limits further biochemical purifi cation . Another approach is to remove Mg 2+ from the buffers and instead substitute the polyamines spermine and spermidine . Alternatively, when purifying nucleoli, we simply removed Mg 2+ from the buffers for a few minutes and then added it back after the initial purifi ca-tion of the nucleoli by centrifugation [ 1 ]. Other methods use low concentrations of nonionic detergents such as Triton X-100 . This has the advantage of dissolving chloroplasts and mitochondria , but the disadvantage of removing the nuclear membranes and mem-brane proteins. The addition of 1 % (v/v) thiodiglycol and 1 M hexylene glycol is sometimes used to improve the stability of nuclei.

Fig. 2 Preparation of nucleoli from Arabidopsis . ( a ) Protoplasts prepared from Arabidopsis culture cells. Bar = 10 μm. ( b ) Purifi ed nuclei prepared from the protoplasts. Bar = 5 μm. ( c ) Arabidopsis nucleoli. Bar = 2 μm


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2 Materials

1. Sterile 10 cm square Petri dishes for plant growth media. 2. Murashige and Skoog (M and S) medium: 0.025 mg/L

CoCl 2 .6H 2 O, 0.025 mg/L CuSO 4 .5H 2 O, 36.7 mg/L Na Fe-EDTA, 6.2 mg/L H 3 BO 3 , 0.83 mg/L KI, 16.9 mg/L MnSO 4 .2H 2 O, 0.25 mg/L Na 2 MoO.2H 2 O, 8.6 mg/l ZnSO 4 .7H 2 O, 332.02 mg/L CaCl 2 .2H 2 O, 170 mg/L KH 2 PO 4 , 1900 mg/L KNO 3 , 180.5 mg/L MgSO 4. 7H 2 O, 1650 mg/L NH 4 NO 3 , pH 5.8, 1 % (w/v) sucrose , 0.5 % (w/v) Phytagel.

3. 10 % (v/v) bleach (contains 5–10 % sodium hypochlorite) in dH 2 O.

4. Arabidopsis , pea, or wheat seeds. 5. Sterile 9 cm round petri dishes and 9 cm round fi lter paper.

1. AT medium: 4.4 % (w/v) M and S medium including vitamins, 3 % (w/v) sucrose, 0.05 mg/L kinetin, and 0.5 mg/L NAA (naphthalene-acetic acid), pH 5.8.

2. ATN medium: 4.4 % (w/v) M & S medium including vitamins, Gamborg B5 vitamins and salts, 3 % (w/v) sucrose, 1 μg/mL 2,4-D (2,4-dichlorophenoxyacetic acid), pH 5.7.

3. Orbital incubator at 150 rpm at 25 °C in dark.

1. Protoplast buffer: 0.5 M sorbitol , 10 mM 2- N -morpholino- ethane-sulphonic acid (MES)-KOH, pH 5.5, 1 mM CaCl 2 .

2. Flotation Buffer: 60 % (v/v) Percoll , 0.5 M sorbitol, 10 mM MES-KOH, pH 5.5, 1 mM CaCl 2 .

3. Nuclei Isolation Buffer (NIB): 10 mM MES–KOH, pH 5.5, 0.2 M sucrose , 2.5 mM EDTA , 2.5 mM DTT, 10 mM NaCl, 10 mM KCl, 0.1 mM spermine , 0.5 mM spermidine , EDTA- free Protease Inhibitor Cocktail Tablets (Roche), containing benzami-dine HCl, phenanthroline , aprotinin , leupeptin , pepstatin A , and phenyl methyl sulfonyl fl uoride (PMSF) are added at ratio of 1 tablet per 50 ml (according to the manufacturer’s instructions).

4. Nucleolar Storage Buffer: 0.35 M sucrose, 0.5 mM MgCl 2 . 5. Protoplasting enzymes: Cellulase “Onozuka” R-10 (Yakult

Pharmaceuticals); Pectolyase Y-23 (MP Biomedicals). 6. Stainless steel homogenizer. We use a plunger type homoge-

nizer, with a spherical ball plunger 25 mm in diameter, in a cylindrical container. The clearance between the plunger and the container walls is approximately 25 μm ( see Note 1 ).

7. Phase contrast microscope. This should be convenient, easy to use, and easily accessible, since it is important to monitor the stages by microscopy at frequent intervals. We use a Zeiss Axiovert 25, with nonimmersion objectives at 5×, 10×, 20×, and 40 × .

2.1 Plant Seedling Growth

2.2 Plant Culture Cell Growth

2.3 Nuclei and Nucleoli Preparation from Culture Cells


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8. Neubauer Hemocytometer counting chamber. 9. Centrifuge . It is important to use a swing-out cooled (bench-

top) centrifuge for all stages of this preparation such as an Eppendorf 5810 R.

1. Microscope slides with frosted end. 2. Shandon Cytospin 4 Cytocentrifuge (Thermo Scientifi c). 3. Shandon Single White Cytofunnels (Thermo Scientifi c). 4. Shandon Cytoclips (Thermo Scientifi c).

1. Paraformaldehyde prilled, store at 4 °C. 2. Triton X-100 . Prepare a stock of 10 % (v/v) Triton X-100 in

dH 2 O and store at 4 °C. 3. Dilute sulfuric acid. Prepare a solution of 10 % (v/v) sulfuric

acid by the careful drop-wise addition of concentrated (98 % (v/v)) sulfuric acid to dH 2 O.

4. Whatman pH 4.5–10 indicator strips. 5. PBS, pH 7.0. 10× stock diluted to 1× for use. 6. NIB with Triton: 10 mM MES-KOH pH 5.5, 0.2 M sucrose ,

2.5 mM EDTA , 2.5 mM DTT, 10 mM NaCl, 10 mM KCl, 0.1 mM spermine , 0.5 mM spermidine , 0.5 % (v/v) Triton X-100.

7. Flat-ended stainless steel rod (140 mm × 3 mm) and/or stain-less steel grinder for 1.5 ml microfuge tube ( see Note 2 ).

8. Nylon mesh fi lter, either CellTrics disposable 30 μm fi lter (Partec) or homemade ( see Note 3 ).

9. Blocking solution: 3 % (w/v) bovine serum albumin (BSA) in PBS, pH 7.0. Make fresh each time.

10. Homemade plastic cover slips made from autoclave bags. 11. 4′, 6-Diamidino-2-phenylindole, DAPI 1 μg/mL solution in

dH 2 O. Protect from light and store at 4 °C. 12. 2, 2′-Thiodiethanol (TDE): 97 % TDE (v/v) 3 % PBS, pH 7.0.

Store at 4 °C and protect from light. 13. Cover slips. Carl Zeiss High Performance cover slips No 1.5.

3 Methods

Carry out all procedures at room temperature unless otherwise stated.

1. Prepare M & S media plates using M & S medium supple-mented with 0.5 % (w/v) Phytagel and 1 % (w/v) sucrose; autoclave for 20 min at 120 °C; allow to cool to about 60 °C before pouring into 9 cm petri dishes while still molten ( see Note 4 ). Allow to cool and solidify before use.

2.4 Cytofunnel Preparation

2.5 Nuclei Preparation for Immunocytochemistry

3.1 Preparation of Arabidopsis Seedlings


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2. Surface sterilize Arabidopsis thaliana seeds in 10 % (v/v) bleach for 10 min, then wash with three changes of sterile water.

3. Plate out individual Arabidopsis thaliana seeds at 2–3 mm spacing in two rows across prepared petri dishes of medium, allowing space for root growth.

4. Stratify the seeds by incubating for 2 days at 4 °C ( see Note 5 ). 5. Germinate and grow Arabidopsis seedlings by placing plates

vertically ( see Note 6 ) in a 25 °C growth chamber under con-stant illumination ( see Note 7 ). Use approximately 5-day- old seedlings for preparation of nuclei.

1. Surface sterilize pea or wheat seeds in 10 % (v/v) bleach for 10 min, then wash with three changes of sterile water.

2. Place pea or wheat seeds on wet fi lter paper in 9 cm petri dishes. 3. Germinate pea and wheat seeds at 20–25 °C in the dark, making

sure the fi lter paper remains wet at all times. Once 4-5 mm of the root tip has emerged it can be used for nuclei preparation.

1. Cut root tips (up to 10 mm in length) ( see Note 8 ) from ger-minated wheat or pea seeds or from Arabidopsis seedlings while still on plates. Collect 10–15 root tips from wheat or pea, or 50–100 root tips from Arabidopsis seedlings and place into 20 mL of fi xative in a 30 mL universal bottle.

2. Vacuum infi ltrate the fi xative until the pieces of root sink in the solution in the absence of a vacuum.

3. Incubate in the fi xative for 1 h. 4. Wash roots in PBS, pH 7.0, 3 × 10 min. 5. Place washed roots into 300-400 μL of NIB + 0.1 % (v/v)

Triton X-100 in a 30 mm glass embryo dish and macerate roots vigorously with a fl at-ended stainless steel rod ( see Note 9 ). Continue macerating for several minutes until the roots have been reduced to tiny pieces releasing the nuclei into the NIB. Alternatively nuclei can be extracted by placing the fi xed roots into a 1.5 mL microfuge tube with the NIB and then a stainless steel grinder can be used to grind the material to release the nuclei.

6. Filter the nuclei solution through a 20 μm nylon mesh fi lter ( see Note 10 ).

7. Either collect the nuclei for biochemistry techniques by cen-trifugation or use for microscopy as detailed as follows.

Different Arabidopsis culture lines require different conditions. The cultures we have used are maintained as follows, but use whatever conditions are recommended for the cell culture that you will use.

3.2 Preparation of Pea or Wheat Seedlings (Or Other Species)

3.3 Isolation of Nuclei from Root Tips

3.4 Maintenance of Arabidopsis Culture Cells


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1. We maintain an Arabidopsis thaliana , Colombia-0 cell culture line by growing in the dark on an orbital incubator at 150 rpm at 25 °C in ATN medium (50 mL of culture per 250 mL coni-cal fl ask).

2. Subculture the cells weekly by diluting 15 mL of culture into 35 mL of fresh ATN medium.

3. We maintain an Arabidopsis thaliana , Landsberg cell culture line by growing in full light at 150 rpm at 25 °C in AT medium (100 mL of culture per 250 mL conical fl ask). Subculture the cells weekly by diluting 6–7 mL into 100 mL fresh AT medium.

1. Harvest the cells from a 3–4-day-old Arabidopsis cell culture by centrifuging at 134 × g for 5 min at room temperature.

2. Gently resuspend the pellet in half the original cell culture vol-ume containing 2 % (w/v) cellulase R-10 and 0.04 % (w/v) pectolyase Y-23 dissolved in protoplast buffer. The cell suspen-sion is gently shaken at 25 °C until most cells are judged by optical microscopy to have formed separated, smooth, round protoplasts, typically 1.5–2 h ( see Notes 11 and 12 ).

3. Harvest the protoplasts by gently centrifuging at 134 × g for 5 min.

4. Resuspend the protoplasts in fl otation buffer using 20 mL per 50 mL of initial cell culture.

5. Overlay the protoplast suspension with a Percoll step gradient . Each 20 ml aliquot of protoplasts in fl otation buffer is over-layed with 5 ml of 45 % (v/v), 5 ml of 35 % (v/v), and 5 ml of 0 % (v/v) Percoll. Percoll solutions are made by diluting fl ota-tion buffer with protoplast buffer to maintain the same osmo-larity ( see Note 13 ).

6. Centrifuge at 134 × g for 5 min and the intact protoplasts will fl oat to the 35–0 % interface.

7. Remove the protoplasts with a Pasteur pipette. This and all subsequent stages and centrifugation steps are carried out on ice or at 4 °C.

8. Wash protoplasts by resuspending in 20 mL protoplast buffer per 50 mL original culture used and recentrifuging at 134 × g for 5 min.

9. Resuspend protoplasts in the same volume of protoplast buffer and count by microscopy using a hemocytometer.

10. Spin down protoplasts as before and resuspend in NIB to give no more than 1 × 10 6 protoplasts per mL of NIB ( see Note 14 )

11. Leave for 5–10 min, then homogenize with one stroke in a stainless steel homogenizer. Check by microscopy that the majority of protoplasts have been ruptured to release nuclei. If necessary, use more strokes in the homogenizer. Spin nuclei

3.5 Isolation of Nuclei from Culture Cells


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down at 209 × g for 5 min and resuspend in NIB ( see Note 15 ). At this stage, nuclei can either be used for isolation of nucleoli (Subheading 3.6 ), for immunolabeling (Subheading 3.7 ) or can be pelleted by centrifugation and used for biochemistry.

1. Homogenize the nuclei further in NIB with more strokes in the stainless steel homogenizer to rupture the nuclei and release nucleoli.

2. When most nuclei have been ruptured, as judged by phase contrast microscopy , spin the nucleoli to pellet at 200 × g , resuspend in Nucleolar Storage Buffer, and freeze in aliquots at −80 °C ( see Notes 16 and 17 ).

1. Pipette 50 μL of nuclei in NIB into each assembled cytofunnel and spin in the Cytospin Cytocentrifuge at 30 × g for 3 min.

2. Disassemble the cytofunnel units, remove the slides, and allow them to air-dry for 40–50 min.

3. Immerse the slides in 70 % (v/v) ethanol for 30 min. 4. Wash with PBS, pH 7.0 for 3 × 10 min. 5. Block tissue with 3 % (w/v) BSA in PBS, pH 7.0 for 1 h ( see

Note 18 ). 6. Apply primary antibodies diluted appropriately in blocking

solution (3 % (w/v) BSA in PBS, pH 7.0) and incubate for a minimum of 2 h at room temperature or overnight at 4 °C.

7. Wash with PBS, pH 7.0 for 6 × 10 min. 8. Apply appropriate secondary antibodies diluted in blocking

solution and incubate for 2 h at room temperature. 9. Wash with PBS, pH 7.0 for 6 × 10 min. 10. Counter-stain for DNA with a 1 μg/mL solution of DAPI in

H 2 O for 30 min. 11. Wash with PBS, pH 7.0 2 × 10 min. 12. Remove as much liquid as possible and add 10–15 μL of a suit-

able mounting media ( see Note 19 ) and cover with a glass cover slip ( see Note 20 ).

13. Seal the cover slip to the slide with nail varnish. 14. View samples with a suitable microscope.

1. To prepare a solution of 4 % (w/v) formaldehyde in PBS, pH 7.0, fi rst prepare a solution of 8 % (w/v) formaldehyde in dH 2 O ( see Note 21 ). Add paraformaldehyde to dH 2 O on a heated stirrer in a fume cupboard. Warm to approx 60 °C and make alkaline by the addition of a few drops of 1 M NaOH. The paraformaldehyde should dissolve to give a clear solution of 8 % (w/v) formaldehyde.

3.6 Isolation of Nucleoli

3.7 Immunofl uore-scence Labeling of Nuclei

3.8 Preparation of Fixative


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2. Add an equal volume of 2× PBS, pH 7.0. This will give a fi nal concentration of 4 % formaldehyde in 1× PBS, pH 7.0.

3. Adjust the pH to 7.0 using dilute H 2 SO4 ( see Note 22 ). 4. Add Triton X-100 to 0.01 %.

1. Place a plain glass slide with frosted end into the cytoclip, keeping the frosted end to the outside of the clip.

2. Position a single white cytofunnel over the slide and secure with the cytoclip. Label appropriately.

4 Notes

1. The construction of the stainless steel homogenizer is as follows (Fig. 3 ). The body of the homogenizer is machined from a solid stainless steel rod (50 mm × 210 mm) by drilling a cylinder into the rod to form a hole of approximately diameter 25 mm and length 200 mm. The top end of the hole is widened by drilling again to increase the opening to approximately width of 38 mm and depth of about 55 mm. The inside of the homogenizer is then fi nished to give a smooth parallel surface, critical for its operation, by either reaming or honing. The plunger to fi t inside the homogenizer is constructed in four pieces. The shaft, the

3.9 Assembly of Cytofunnel Unit

Fig. 3 Stainless Steel homogenizer. ( see Note 1 ) Bar in ( a ) = 50 mm


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ball end, the handle, and the cap. The shaft is made from a 265 mm × 10 mm piece of stainless steel rod with a 10 mm thread (7 mm in length) machined on each end for attachment of the ball end and the handle. The ball end is machined from a suitable width stainless steel rod to form a sphere, profi led to match the drilled shaft on the inside of the homogenizer and to give a clearance of 25 μm. It is drilled on one end to accommo-date the screw thread of the shaft. The shaft handle can be shaped as wished and drilled in the center to accept the 10 mm thread of the shaft. The cap is machined from Acetal rod, drilled in the center to accept the shaft, and shaped to cover the end of the homogenizer to prevent any splashes during operation. A 2 mm diameter pressure release hole is drilled through the cap.

2. The stainless steel maceration rod was made by cutting a 140 mm length of 3 mm stainless steel rod and removing any sharp edges by gently grinding the cut edges to leave a fl at- bottomed rod (Fig. 4 ). The grinder to fi t a microfuge tube was turned from a 20 mm × 8 mm piece of stainless steel rod to give the internal shape of a microfuge tube (10° angle) with the end rounded to fi t the bottom of the tube. This was screw tapped to accept the 5 mm stem also made from stainless steel which is screwed into the head. A plastic handle can be added for comfort.

Fig. 4 Maceration rod and grinder ( see Note 2 ). ( a ) Stainless steel rod with fl at end. ( b ) Stainless steel grinder made to the internal profi le of a microfuge tube ( c )


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3. A homemade fi lter can be made from the body of a 20 mL syringe with the tip cut off, the cut end is then covered with a piece of 30 μm nylon net fi lter (Millipore) and secured with tape.

4. It is not possible to remelt phytagel so allow the medium to cool to a reasonable working temperature of about 60 °C before pouring the plates under sterile conditions.

5. Stratifi cation of seeds by a cold treatment of 2 days will ensure an even germination rate.

6. By placing the plates vertically, the germinating roots will grow along the surface of the gel and can be removed easily without damage to the root structure .

7. We use 25 °C, constant light growth conditions to germinate Arabidopsis seedlings . However, this procedure works equally well with seedlings grown at cooler temperatures with light/dark cycles. It should be noted that different conditions need different times to reach the same stage of development.

8. Using the fi rst 10 mm of the root tip will ensure the availability of nuclei from both meristematic and differentiated tissue.

9. The maceration step can take several minutes of continuous stabbing with the fl attened end of the stainless steel rod to effectively release a substantial amount of nuclei. A good guide is to reach a point when there are very small pieces of root remaining and the solution is partially cloudy.

10. The nylon mesh fi lter should be wetted with NIB prior to use. 11. Harvested cells are resuspended in solutions of wall-degrading

enzymes and gently shaken until most cells are visible as round protoplasts detached from the cells around them. Ensuring good quality protoplasts at this stage is vital for the purifi cation procedure. The protoplasting stage is sensitive to changes in temperature, and excessive enzyme treatment tends to lead to highly unstable protoplasts.

12. Arabidopsis culture cells are rather more amenable to proto-plasting than some species, but seem to have a strong require-ment for pectolyase. Indeed, the concentration and condition of this enzyme appears to be rate determining, as the use of a different stock from the same supplier produced unstable pro-toplasts. Pectolyase is responsible for cleaving the pectin oligo-saccharides that ‘cement’ the cellulose fi brils into the wall structure suggesting that digestion of the wall with cellulase alone is not very effective if the pectin matrix is still holding the fragments in place.

13. The cell culture protoplasts are purifi ed on a discontinuous Percoll gradient. This is necessary to remove contamination from protoplasts that have lysed during enzymatic treatment or the remains of dead cells from the original culture. The use of Percoll


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gradients can lead to problems, however, as the exact conditions used can prove sensitive. For example, an extraction of nucleoli from cells grown and extracted not in the usual AT medium and resuspension buffers but in a sucrose-free minimal medium altered the osmolarity of the original protoplast suspension so as to cause the protoplasts to simply pellet, along with other cellular material. Hence, only a partial purifi cation was achieved, and the sample required additional centrifugal purifi cations. On the other hand, protoplasts grown in sucrose were observed to migrate on the Percoll gradient much more cleanly, suggesting that a small increase in osmolarity can aid extraction [ 2 ].

14. Once further cleaned by centrifugation, the harvested proto-plasts are used for the extraction of nuclei. This method uses hypertonic disruption of protoplasts in NIB [ 4 ]. The various elements of this mixture were empirically determined to improve stability and yield. The polyamines spermine and sper-midine , and DTT stabilize chromatin; EDTA disrupts chroma-tin by chelating Mg 2+ , but inhibits phenol oxidases and DNAses; polyamines prevent EDTA-mediated chromatin disruption.

15. Protoplasts are disrupted by a stainless steel homogenizer, releasing nuclei. If necessary, the nuclei can be purifi ed by fi l-tering through layers of Miracloth and mesh fi lters, but we do not usually fi nd this necessary. If required, suitable fi lters are available (such as Millipore Nylon Net Filters, available in 11, 20, 30, 40, 60 μm). Start with a coarse fi lter (e.g., 30 μm), fi nishing with a fi ne fi lter (e.g., 11 μm)—depending on the size of the nuclei.

16. Control of the Mg 2+ concentration is important in isolating nucleoli, if Mg 2+ is added to the NIB, the nucleoli cannot be separated from the network of nuclear chromatin fi bers, whereas without Mg 2+ in the buffer the nucleoli begin to show signs of disintegration after 1–2 h. Therefore, Mg 2+ is added to the storage buffer, within 30 min of nuclear breakage.

17. Numbers of nuclei/nucleoli extracted vary with the age of cells used. Cultures have been used between 18 h and 10 days after subculturing, although most have been 3–4 days old. Typically, 5 × 10 6 –5 × 10 7 nucleoli are extracted from 100 mL of initial culture. In a published study using this method, 1 L of cell culture generated 4.8 × 10 8 nuclei and subsequently 3.2 × 10 8 nucleoli [ 4 ].

18. To ensure the blocking and labeling solutions keep in contact with the sample, either a plastic cover slip can be placed over the sample and solution or a temporary well can be made by using a Pap pen to draw a well around the sample area.

19. There are many mounting solutions available, but, as it is important for optimal image collection to match refractive


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indices as closely as possible within your imaging setup, careful consideration of the mounting medium is essential. Ideally the refractive index of the immersion medium for the lens (usually oil), the glass cover slip and the sample mounting medium should be as closest to each other as possible. The mounting medium should also have good antifade properties and be able to limit the amount of fl uorescence quenching through photo-bleaching. We have found that a solution of 97 % (v/v) TDE in PBS, pH 7.0 [ 6 ] meets these requirements optimally.

20. Most objectives designed for use in high resolution biological imaging are calculated for a cover slip thickness of 170 nm (No 1.5). For the highest quality imaging we recommend using high-precision cover slips such as Carl Zeiss high performance cover slips, as these have a much smaller deviation from the nominal 170 nm than standard cover slips.

21. We fi nd it best to make fresh formaldehyde each time. Formaldehyde is a carcinogen so should always be used in a fume cupboard. Weigh out paraformaldehyde in the fume cup-board, wearing appropriate safety clothing, lab coat, gloves, and eye protection. Warm the solution but do not allow it to boil, as this will degrade the formaldehyde. We recommend using paraformaldehyde “prilled” rather than powder to avoid harmful dust. Paraformaldehyde dissolves best at alkaline pH. Therefore, it is best to make it in H 2 O with a few drops of alkali (rather than in a buffer) and then add a 2× buffer once the formaldehyde is dissolved. In this way, less acid is needed to bring the pH back to neutral. If more than a few drops of alkali are needed to dissolve the paraformaldehyde, this is probably a sign that the paraformaldehyde has degraded and should be replaced. Paraformaldehyde should be kept dry at all times. It lasts longer at 4 °C than at room temperature but should be allowed to warm to room temperature before open-ing the container to avoid condensation.

22. To adjust the pH of formaldehyde, do NOT use HCl, as reac-tion of formaldehyde and HCl produces the carcinogen Bis (chloromethyl) ether. Also use pH strips to determine the pH rather than a pH electrode, as fi xatives can degrade pH electrodes.

Acknowledgements

This work was supported by grant BB/J004588/1 from BBSRC and the John Innes Foundation.


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References

1. Pendle AF, Clark GP, Boon R et al (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 16:260–269

2. McKeown P, Pendle AF, Shaw PJ (2008) Preparation of Arabidopsis nuclei and nucleoli. Methods Mol Biol 463:67–75

3. Pendle AF, Shaw PJ (2015) Immunolabelling and in situ labelling of isolated plant interphase nuclei. Methods Mol Biol. In press

4. Saxena PK, Fowke LC, King J (1985) An effi -cent procedure for the isolation of nuclei from plant protoplasts. Protoplasma 128:184–189

5. Jackson DA, Cook PR (1985) A general method for preparing chromatin containing intact DNA. EMBO J 4:913–918

6. Staudt T, Lang MC, Medda R et al (2007) 2,2’-Thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc Res Tech 70:1–9


chapter 3 · concentrations of nonionic detergents such as triton x-100 . ... less steel grinder...

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