We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. Chromosome Preparation
<ol>
	<li>Seed germination and fixation of root tips
	<ol>
		<li>Germinate 10-20 barley seeds on two layers of moist filter paper in a Petri dish under dark conditions for 2 days at 22-24 &#176;C. Cut off vigorous roots with the length of 1-2 cm from the seed by using a razor blade.</li>
		<li>Prepare ice-cold water by placing a 500 ml glass bottle containing cold tap water into crushed ice-water. Aerate the ice-cold water and immerse root tips for 20 hr to increase the frequency of metaphase cells.</li>
		<li>Transfer roots from water to 50 ml of ethanol: acetic acid (3:1) fixative to fix them at RT&#160;for 2 days. Store roots in a freshly prepared ethanol: acetic acid (3:1) fixative at 4 &#176;C until use up to a year.</li>
	</ol>
	</li>
	<li>Washing and enzyme treatment
	<ol>
		<li>Wash the 10-20 roots with 30 ml ice-cold tap water for 5 min twice using a 50 ml glass beaker. Use binocular microscope. Transfer roots one by one into 30 ml 0.01 M citrate buffer (0.01 M citric acid + 0.01 M sodium citrate, pH 4.8) using forceps and wash by shaking the glass beaker for 5 min twice. Place roots on filter paper to remove the liquid completely and cut-off undesired non-meristematic tissue using a razor blade.</li>
		<li>Incubate up to 20 root tips in 1 ml enzyme mixture at 37 &#176;C for about 50 min to soften the plant tissue (Table 1) in a watch glass. Enzyme mixture contains 0.7% cellulase R10, 0.7% cellulase, 1% pectolyase and 1% cytohelicase diluted in 0.01 M citrate buffer. Store the enzyme mixture at -20 &#176;C and reused up to five times.</li>
		<li>Remove the enzyme by pipetting and wash the root tips on ice with 5 ml 0.01 M citrate buffer twice to replace the residual enzyme.</li>
	</ol>
	</li>
	<li>Root maceration
	<ol>
		<li>Wash root tips with 1 ml 96% ethanol twice carefully in the same watch glass. Replace ethanol with freshly prepared fixative (75% acetic acid : 25% ethanol). Use 10-15 &#181;l fixative per root tip.</li>
		<li>Transfer root tips together with the fixative into a 2 ml tube and disintegrate root meristems with a dissecting needle or forceps. Tap the tube 20 times to re-suspend cells to obtain a cell suspension. Store the cell suspension at -20 &#176;C up to two months.</li>
	</ol>
	</li>
	<li>Dropping of the cell suspension
	<ol>
		<li>Place 2-3 layers of water-soaked paper tissue on a hot plate at 50 &#176;C. Immerse microscopic slides in ice-cold tap water in the fridge for 30 min. And place slides on top of the moist paper tissue.</li>
		<li>Pipette 7-10 &#181;l of cell suspension and drop it from a distance of 20 cm onto the cooled slide placed on the hot plate. Pipette 10 &#181;l of acetic acid-ethanol mixture on the same place as cell suspension on the slide and keep the slide on the hot plate for additional 2 min. Place the slide on the hot plate without the wet tissue and let it dry for 1 min.</li>
	</ol>
	</li>
	<li>Quality control and storage of slides
	<ol>
		<li>Check slides using a phase-contrast microscope to control the quality of the chromosome spread. Use slides either the same day or store by immersing in 96% ethanol in a Coplin jar at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Pretreatment of slides before FISH; all steps are carried out at RT
	<ol>
		<li>Place slides in a Coplin jar containing 50 ml of 2x SSC (20x SSC contains 3 M NaCl and 300 mM trisodium citrate) for 5 min. Using forceps, transfer slides to a Coplin jar containing 50 ml of 45% acetic acid for 3-10 min.</li>
		<li>Transfer slides to a Coplin jar containing 50 ml of 2x SSC for 10 min. Transfer slides to a Coplin jar containing 50 ml of 4% formaldehyde (in 2x SSC) and immerse slides for 10 min to fix chromosomes.</li>
		<li>Remove formaldehyde by rinsing the slides 3 times for 4 min each, in a Coplin jar containing 50 ml 2x SSC. Dehydrate slides in a Coplin jar for 2 min in series of 70%, 90% and 100% ethanol, respectively and dry slides in a vertical position.</li>
	</ol>
	</li>
</ol>
2. Fluorescent In Situ Hybridization (FISH)
<ol>
	<li>For each slide, prepare a hybridization solution of 20 &#181;l in total using 10 &#181;l of deionized formamide, 5 &#181;l of 4x hybridization buffer (200 &#181;l buffer contains 80 &#181;l 20x SSC, 8 &#181;l 1 M Tris-HCl pH 8.0, 1.6 &#181;l 0.5 M EDTA, 11.2 &#181;l 10 &#181;g/ &#181;l salmon sperm and 99.2 &#181;l DNase-free water), 3 &#181;l of the probe and 2 &#181;l of DNase-free water.</li>
	<li>Add 20 &#181;l of hybridization solution per slide and cover with a 24 x 32 mm cover slip and arrest the cover slip with rubber cement. Denature slides with probes simultaneously at 80 &#176;C for 2 min on a hot plate.</li>
	<li>Transfer slides to a moist chamber and incubate slides at 37 &#176;C O/N avoiding light. Remove cover slips by rinsing the slides in a Coplin jar with 2x SSC. Place slides in a Coplin jar containing 55-60 &#176;C 2x SSC and incubate for 20 min.</li>
	<li>Place the slides to 2x SSC in a Coplin jar for 2 min at RT. Dehydrate slides in a Coplin jar for 2 min in series of 70%, 90% and 100% ethanol, respectively.</li>
	<li>Air-dry the slides and counterstain with 1 &#181;g/ml 4&#39;,6-diamidino-2-phenylindoline (DAPI) in antifade mounting medium, avoid intense light.</li>
</ol>
3. Microscopic Analysis and Storage
<ol>
	<li>Analyze the slides using an epifluorescence microscope. The selection of filter depends on the fluorochrome used for probe labeling. If necessary, store slides at 4 &#176;C under dark conditions up to a year.</li>
</ol>

<ol>
	<li>Geber, G., Schweizer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+heterochromatin+differentiation+in+Sinapis+alba+(Cruciferae)+using+a+simple+air-drying+technique+for+producing+chromosome+spreads.">Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads.</a> Pl Syst Evol. 158 (2-4), 97-106 (1988).</li><li>Andras, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drop-spreading+technique+to+produce+cytoplasm-free+mitotic+preparations+from+plants+with+small+chromosomes.">A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.</a> Chromosome Res. 7 (8), 641-647 (1999).</li><li>Rothfels, K. H., Siminovitch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air-drying+technique+for+flattening+chromosomes+in+mammalian+cells+grown+in+vitro.">An air-drying technique for flattening chromosomes in mammalian cells grown in vitro.</a> Stain Technology. 33 (2), 73-77 (1958).</li><li>Martin, R., Busch, W., Herrmann, R. G., Wanner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+preparation+of+plant+chromosomes+for+high-resolution+scanning+electron+microscopy.">Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy.</a> Chromosome Res. 2 (5), 411-415 (1994).</li><li>Kato, A., Albert, P. S., Vega, J. M., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+fluorescence+in+situ+hybridization+signal+detection+in+maize+using+directly+labeled+probes+produced+by+high+concentration+DNA+polymerase+nick+translation.">Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.</a> Biotech. Histochem. 81 (2-3), 71-78 (2006).</li><li>Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A., Khrustaleva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+'SteamDrop'+method+for+high+quality+plant+chromosome+preparation.">An easy 'SteamDrop' method for high quality plant chromosome preparation.</a> Mol. Cytogenet. 7, 21 (2014).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61 (4), 387-393 (1996).</li><li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synteny+between+Brachypodium+distachyon+and+Hordeum+vulgare+as+revealed+by+FISH.">Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH.</a> Chromosome Res. 18 (7), 841-850 (2010).</li><li>Kato, A., Lamb, J. C., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+painting+using+repetitive+DNA+sequences+as+probes+for+somatic+chromosome+identification+in+maize.">Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.</a> Proc. Natl. Acad. Sci. U S A. 101 (37), 13554-13559 (2004).</li><li>Pan, W. H., Houben, A., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+cell+synchronization+in+plant-roots+by+hydroxyurea+and+amiprophos-methyl+or+colchicine.">Highly effective cell synchronization in plant-roots by hydroxyurea and amiprophos-methyl or colchicine.</a> Genome. 36 (2), 387-390 (1993).</li><li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+karyotyping+of+sorghum+by+in+situ+hybridization+of+landed+BACs.">Integrated karyotyping of sorghum by in situ hybridization of landed BACs.</a> Genome. 45 (2), 402-412 (2002).</li><li>Lapitan, N. L. V., Brown, S. E., Kennard, W., Stephens, J. L., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+physical+mapping+with+barley+BAC+clones.">FISH physical mapping with barley BAC clones.</a> Plant J. 11 (1), 149-156 (1997).</li><li>Aliyeva-Schnorr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+mapping+with+centromeric+BAC+contigs+shows+that+this+recombination-poor+region+comprises+more+than+half+of+barley+chromosome+3H.">Cytogenetic mapping with centromeric BAC contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.</a> Plant J. 84, 385-394 (2015).</li></ol>

The authors have nothing to disclose.

The chromosome preparation experiment has been carried out using young roots of cereals belonging to the grass family (Poaceae). All analyzed species have 14 relatively long mitotic metaphase chromosomes (11-15 &#181;m) in the diploid genome set and belong to large-genome species (5.1-7.9 Gbp).
Length of germinated roots was not more than 2 cm to obtain a maximum of meristematic tissue. Synchronization of dividing cells was achieved by a 20 hr long ice-water treatment that improved the quantity of mitotic metaphase spreads 10.
Two steps are important for the preparation of high-quality chromosome preparations: (I) the relative humidity of 50%-55% and (II) duration of the enzyme treatment. The first point was achieved by placing wet paper tissues on a hot plate in proximity of the glass slides. The relative humidity was measured with a hygrometer. The optimal humidity for the preparation of plant chromosomes was similar to the humidity reported by Kirov et al. 6. The positive effect on the chromosome quality at optimal relative humidity occurs by swelling of the cytoplasm and cell wall hydrolysis.
The duration of enzyme treatment is species dependent (Table 1). The period of enzyme treatment also depends on the time span of root fixation in ethanol/acetic acid and the size of the roots. The longer roots were stored in the fixative (up to 1 year at 4 &#176;C), the longer it takes to digest roots to the proper grade. Insufficiently digested root material is difficult to macerate and will increase the total time of preparation as a result of long lasting maceration. Moreover, metaphase chromosomes remain embedded into cytoplasm that could hamper ensuing probe penetration during the FISH experiment. On the other hand over-digested material can influence the structure of the chromosomes themselves, and damage target DNA for the FISH analysis.
An additional factor for the improvement of the preparation is the use of the second drop of fixative (3:1, acetic acid/ethanol). High concentration of acetic acid in this mixture stimulates the digestion of cytoplasm and promotes chromosome spreading in species with large chromosomes. Cytoplasm reduction can also take place after the immobilization of the chromosomes on slides. For this purpose microscope slides carrying the chromosome spreads can be incubated in 45% acetic acid at RT for 2-10 min depending on cytoplasm level. Quality check of chromosome spreads was performed with a phase-contrast microscope without any supplementary staining (e.g., 1% aceto-carmin). Normally more than 25 slides containing high-quality chromosome spreads can be obtained from 20 roots using the method above.
Results of the FISH experiments on mitotic metaphase chromosomes using 5S rDNA, [CTT]10, and 6 kb long single-copy probe (FPct_40752) were distinct and of a high quality for all species described above (Figure 2). Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. It is possible to store the cell suspension at -20 &#176;C up to two months and to prepare the chromosome spreads on the day of the FISH experiment. Freshly prepared slides can be also stored at -20 &#176;C in 96% ethanol, though we observed that the quality of hybridization signals on such chromosomes is reduced compared to the freshly-prepared metaphase spreads.
Chromosome spreads prepared by the fast air-dry dropping technique were suitable for FISH and were reproduced a number of times. Combination of this chromosome preparation method with FISH could be widely applied to explore the genome organization in plants, for instance, for karyotyping11, chromosomal mapping 12, in synthetic studies, and for the integration of physical and genetic maps13.

Fluorescence in situ hybridization (FISH) is an effective tool for the physical mapping of single and high copy sequences at the chromosomal level. Prerequisite is the preparation of high quality chromosome spreads. There is no general chromosome preparation protocol that would be equally suitable for animal and plant cells. Preparation of plant chromosomes is particularly challenging due to the rigid cell wall and various cytoplasm consistency within different species. One of the favorable methods for the preparation of plant chromosomes is a so-called drop technique also known as drop-spreading technique and air-drying technique 1,2. This method was first introduced in 1958 by Rothfels and Siminovitch for in vitro grown mammalian cells 3. Later Martin et al. 4 and Kato et al. 5 adapted this method for plants.
More recently, a method named &#39;SteamDrop&#39; was developed which used water steam for the preparation of non-overlapping chromosomes 6. Although, the positive influence of high humidity was observed earlier 7, &#39;SteamDrop&#39; delivers a controlled workflow of high-quality chromosome preparations 6. The steam treatment causes stretching of chromosomes probably connected to some modifications of chromosomal proteins. The quality of resulting metaphase spreads is very high, although retaining of sufficient number of complete metaphase spreads for subsequent FISH experiments demands technical expertise.
Here we present a protocol for the preparation of mitotic cereal chromosomes suitable for the FISH detection of single and high copy probes 5,8. This method is an improved variant of the air-dry dropping method described by Kato 9 performed under relative humidity of 50%-55% (Figure 1). This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Using this protocol we obtained high-quality chromosome spreads and FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

<table>
	<tbody>
		<tr>
			<td>Hot Plate</td>
			<td>MEDAX GmbH</td>
			<td>12603</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase R10</td>
			<td>Duchefa</td>
			<td>C8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase&#160;</td>
			<td>CalBioChem</td>
			<td>219466</td>
			<td></td>
		</tr>
		<tr>
			<td>Pectolyase</td>
			<td>Sigma</td>
			<td>P3026</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytohelicase</td>
			<td>Sigma</td>
			<td>C8274</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-12-dUTP</td>
			<td>Invitrogen</td>
			<td>C3176</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluor488-5-dUTP</td>
			<td>Invitrogen</td>
			<td>C11397</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluorecsence microscope</td>
			<td>Olympus BX61</td>
			<td>BX61</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Orca ER, Hamamatsu</td>
			<td>C10600</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>fluorecsent dye</td>
		</tr>
	</tbody>
</table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

Microscopic slides with the mitotic metaphase spreads were prepared by the fast air-dry dropping chromosome preparation method described above (Suppl. Figure 1). FISH analysis was carried out using both, repetitive and single-copy sequences. Images were obtained by a epifluorescence microscope with a set of filters enabling excitation of corresponding fluorophores and captured by a high-sensitivity CCD monochrome camera. For the image acquisition we used a computer with an image acquisition software. Results of the FISH experiments on mitotic metaphase chromosomes using 5S rDNA, [CTT]10, and single-copy probes were distinct and of a high quality for Hordeum vulgare (Figre 2A, B), H. bulbosum (Figure 2C), H. marinum, H. murinum, H. pubiflorum and Secale cereale (Figure 2D). Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. It is possible to store the cell suspension at -20 &#176;C up to two months and to prepare the chromosome spreads on the day of the FISH experiment. Freshly prepared slides can be also stored at -20 &#176;C in 96% ethanol, though we observed that the quality of hybridization signals on such chromosomes is reduced compared to the freshly-prepared metaphase spreads. The methods can be used to prepare high-quality chromosome spreads in cereals in an easy, efficient and reproducible way and most likely can be used in other plant species too.
<img alt="Figure 1" src="/files/ftp_upload/53470/53470fig1.jpg" /> 
Figure 1. A scheme describing the procedure of the air-dry dropping plant chromosome preparation method.
<img alt="Figure 2" src="/files/ftp_upload/53470/53470fig2.jpg" /> 
Figure 2. FISH on mitotic metaphase chromosome spreads of Hordeum vulgare, H. bulbosum and Secale cereale prepared by the air-dry dropping method.&#160;(A)&#160;H. vulgare with a single copy probe (FPct_40752) labeled with a red fluorescent dye. (B)&#160;H. vulgare with 5S rDNA probe labeled by a green fluorescent dye. (C)&#160;H. bulbosum with CTT-microsatellite labeled by a green fluorescent dye and (D)&#160;S. cereale with pSc119.2 repeat labeled by a green fluorescent dye. All chromosomes were counterstained with DAPI (in red). FISH signals are shown in yellow. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53470/53470fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/53470/53470tab1.jpg" />
Table 1. Incubation time of enzyme treatment for different species.
<img alt="Figure 3" src="/files/ftp_upload/53470/53470fig3.jpg" /> 
Suppl. Figure 1. Phase-contrast and differential interference contrast (DIC) images of mitotic metaphase chromosome spreads of the air-dry dropping plant chromosome preparation method on the example of Hordeum vulgare. (A)&#160;Phase-contrast image taken at 200X magnification and (B) Differential interference contrast image taken at 630X magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53470/53470fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

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13.1K Views.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Video: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

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Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

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Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

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Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

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Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Chromosome Preparation From Cultured Cells

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes