This research was funded by the Central Public-interest Scientific Institution Basal Research Fund of State Key Laboratory of Tree Genetics and Breeding (grant number CAFYBB2020ZY001-2), Enterprise research projects(202404016-4320), and National Natural Science Foundation of China (NSFC 32271896).

1. Collection of <img alt="Equation 2" src="/files/ftp_upload/67350/67350eq02.jpg" style="margin: auto;" /> B. xylophilus
<ol>
	<li>Collection of infected disks of P. massoniana 
	NOTE: All procedures complied with the Administrative Measures for Pine Wilt Disease Epidemic Area and Infected Wood and other applicable regulations. A P. massoniana forest infected by B. xylophilus in Xiangshan County, Ningbo City, Zhejiang Province, was selected as the sample site. P. massoniana, which died from pine wilt disease, was felled within the sample site.
	<ol>
		<li>Collect round logs (5 cm thick) from the upper, middle, and lower sections of the main trunk of the felled trees.</li>
		<li>Cut the collected round logs of P. massoniana into 0.5-1 cm thick slices, then trim them into small strips of 2-3 cm length.</li>
		<li>Place the small wood strips into sealed bags and store them in a laboratory for further use.</li>
	</ol>
	</li>
	<li>Extraction of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>Attach a rubber tube to the narrow end of a long-neck glass funnel with an approximate diameter of 15 cm. Secure the rubber tube with a pinchcock. Place the assembled funnel on a funnel stand.</li>
		<li>Wrap the small wood strips completely in a single layer of tissue paper and then place them in the funnel. Add distilled water to the funnel until all wood strips are submerged. 
		&#8203;NOTE: After standing for 12-24 h, The nematodes migrated out of the wood strips and settled at the bottom of the funnel10. Tissue paper prevented wood debris from settling while allowing nematodes to pass through.</li>
		<li>Release the pinchcock at the bottom of the funnel slowly, and let 2000-5000 &#181;L of the mixture leak into a Petri dish (5 cm in diameter) for microscopic examination and morphological identification.</li>
		<li>Identify <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus by observing features such as stylets, median esophageal bulbs, lipid droplets, and &#34;finger-like&#34; tail shapes11.</li>
		<li>Under a microscope, transfer individual <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus onto a slide by aspirating 10 &#181;L of liquid along with one nematode using a 10 &#181;L pipette each time. Repeat this process 30 times to collect approximately 300 &#181;L of the mixture containing nematodes.</li>
		<li>After extraction of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, sterilize the remaining wood strips and infected wood materials to prevent further contamination.</li>
	</ol>
	</li>
</ol>
2. Osmotic regulation process control
<ol>
	<li>Preparation of osmotic regulation mixture
	<ol>
		<li>Prepare an 8% KCl aqueous solution.</li>
		<li>Using a pipette, add 10 &#181;L of the 8% KCl aqueous solution to the slide containing the mixture of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus, yielding approximately 310 &#181;L of a mixture of the nematodes and KCl solution.</li>
	</ol>
	</li>
	<li>Regulation of increased osmotic pressure and dehydration of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus&#8203;
	<ol>
		<li>Place the slide on the microscope stage, allowing the water to evaporate naturally. It would gradually increase the osmotic pressure of the surrounding environment, causing nematodes to dehydrate over approximately 30-60 min. 
		NOTE: The operator only needs to wait for the water to evaporate naturally until the KCL crystals precipitate and observe the state of the nematodes under a microscope. It takes about 30-60 min for 310 &#181;L of the mixture to evaporate naturally until the KCL crystals precipitate.</li>
		<li>Observe the dehydration process (e.g., body curling, aggregation, etc.) of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus under the microscope. When the water fully evaporates and KCl crystals precipitate, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus stops moving and enters cryptobiosis.</li>
	</ol>
	</li>
	<li>Low-temperature stress treatment of cryptobiotic <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>Place the cryptobiotic <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus in a constant temperature environment of -20 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Regulation of decreased osmotic pressure and rehydration of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus
	<ol>
		<li>Add approximately 300 &#181;L of distilled water around the juveniles that had undergone low-temperature treatment by pipette. The rehydration process, lasting for about 30-60 min, gradually decreases the osmotic pressure of their surrounding environment.</li>
		<li>Observe the rehydration process of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus (e.g., body stretching, resumption of movement, etc.) under the microscope. Calculate the survival rate after 24 h.</li>
	</ol>
	</li>
</ol>

Cryptobiosis of Bursaphelenchus xylophilus Juveniles

<ol>
	<li>Li, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+on+the+trend+of+invasion+and+expansion+of+Bursaphelenchus+xylophilus.">Analysis on the trend of invasion and expansion of Bursaphelenchus xylophilus.</a> Forest Pest and Disease. 37 (5), 1-4 (2018).</li><li>Futai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pine+wood+nematode,+Bursaphelenchus+xylophilus.">Pine wood nematode, Bursaphelenchus xylophilus.</a> Annu Rev Phytopathol. 51 (51), ?61 (2013).</li><li>Crowe, J. H., Cooper, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptobiosis.">Cryptobiosis.</a> Sci Am. 225, 30-36 (1971).</li><li>Wharton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis.">Anhydrobiosis.</a> Curr Biol. 25, 1107-1125 (2015).</li><li>Browne, J., Tunnacliffe, A., Burnell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis:+plant+desiccation+gene+found+in+a+nematode.">Anhydrobiosis: plant desiccation gene found in a nematode.</a> Nature. 416 (6876), 38 (2002).</li><li>Shatilovich, A. V., 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=Viable+nematodes+from+late+Pleistocene+permafrost+of+the+Kolyma+river+lowland.">Viable nematodes from late Pleistocene permafrost of the Kolyma river lowland.</a> Dokl Biol Sci. 480 (1), 100-102 (2018).</li><li>Schottenfeld-Roames, J., Ghabrial, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+regulation+of+seamless+tube+growth.">Osmotic regulation of seamless tube growth.</a> Nat Cell Biol. 15 (2), 137-139 (2013).</li><li>Erkut, 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=Trehalose+renders+the+dauer+larva+of+Caenorhabditis+elegans+resistant+to+extreme+desiccation.">Trehalose renders the dauer larva of Caenorhabditis elegans resistant to extreme desiccation.</a> Curr Biol. 21 (1), 1331-1336 (2012).</li><li>Pan, 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=Third-stage+dispersal+juveniles+of+Bursaphelenchus+xylophilus+can+resist+low-temperature+stress+by+entering+cryptobiosis.">Third-stage dispersal juveniles of Bursaphelenchus xylophilus can resist low-temperature stress by entering cryptobiosis.</a> Biology. 10 (8), 785 (2021).</li><li>Mao, X. F., Li, H. X., Chen, X. Y., Hu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+efficiency+of+soil+nematodes+by+different+methods.">Extraction efficiency of soil nematodes by different methods.</a> Chinese J Ecol. 23 (3), 149-151 (2004).</li><li>Pan, 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=Isolation+and+identification+of+pine+wood+nematode+in+Pinus+koraiensis+in+Fengcheng,+Liaoning+province.">Isolation and identification of pine wood nematode in Pinus koraiensis in Fengcheng, Liaoning province.</a> Forest Pest and Disease. 38 (1), 4 (2019).</li><li>Pan, L., Cui, R., Li, Y., Feng, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+pinewood+nematodes+in+Pinus+tabuliformis+Carr.+under+low-temperature+conditions+in+Fushun,+China.">Investigation of pinewood nematodes in Pinus tabuliformis Carr. under low-temperature conditions in Fushun, China.</a> Forests. 11 (9), 993 (2020).</li><li>Madin, K. A. C., Crowe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis+in+nematodes:+Carbohydrate+and+lipid+metabolism+during+dehydration.">Anhydrobiosis in nematodes: Carbohydrate and lipid metabolism during dehydration.</a> J Exp Zool. 3, 193 (2010).</li><li>Wharton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis:+The+model+worm+as+a+model.">Anhydrobiosis: The model worm as a model.</a> Curr Biol. 21 (15), 578-579 (2011).</li><li>Li, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+risk+of+invasion+and+expansion+of+pine+wood+nematode+in+the+middle+temperate+zone+of+China.">High risk of invasion and expansion of pine wood nematode in the middle temperate zone of China.</a> J Temperate Res. 1 (1), 4 (2018).</li></ol>

The authors have nothing to disclose.

In this study, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus was collected from dead P. massoniana in Ningbo, Zhejiang Province. It should be noted that <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus was selected from a mixed population with different instars. Specifically, round logs with a high density of B. xylophilus were chosen, followed by the screening of <img alt...

Pine wilt disease is one of the most severe forest diseases in China1. The pinewood nematode Bursaphelenchus xylophilus (B. xylophilus) poses a significant threat to pine species such as Pinus massoniana (P. massoniana) by reproducing within the host, leading to rapid wilting and death. Under unfavorable environmental conditions after the death of the host, the reproductive juveniles of B. xylophilus may enter pre-dauer juveniles (<img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" />). These juveniles seek insect vectors to continue their transfer and spread, causing extensive pine forest die-offs2.
Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress3. In this process, organisms gradually dehydrate, resulting in halted movement, reduced metabolism, and a suspended lifecycle4. Under suitable conditions, they can recover upon rehydration. Cryptobiosis enables organisms to resist extreme environments, such as low temperatures, and extend their lifespans5. Nematodes that survived in cryptobiosis for 30,000 years were found in Antarctic permafrost6.
Previous studies have shown that nematodes can adapt to various abiotic stressors -- such as low temperature, drought, and high osmotic pressure -- during dehydration by adjusting their surface permeability7. Additionally, nematodes can curl their bodies to decrease surface area and minimize water loss rate. For example, Caenorhabditis elegans regulates osmotic balance, allowing ions and small molecules to freely pass through its cuticle, while larger molecules cannot8. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus also exhibits cryptobiosis. During the dehydration and rehydration stages of cryptobiosis, osmotic regulation is essential for controlling substance exchange across the cuticle, making it pivotal for entering or recovering from cryptobiosis9.
The present study introduces a method for inducing cryptobiosis in <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus through osmotic regulation. This method is simple, efficient, and highly reliable, providing technical support for researching the stress resistance mechanisms of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass slide</td>
			<td>Bioland</td>
			<td>GS7101-7525</td>
			<td>75 mm &#215; 25 mm &#215; 1 mm</td>
		</tr>
		<tr>
			<td>Long necked glass funnel</td>
			<td>Nanjing Kangluoda Experimental Technology</td>
			<td>N/A</td>
			<td>150 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Phenix</td>
			<td>XSP-35-1600X</td>
			<td>PH100-2B41L-IPL</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Nanjing Kangluoda Experimental Technology</td>
			<td>&#160;TB-624935792745</td>
			<td>50 mm &#215; 50 mm</td>
		</tr>
		<tr>
			<td>Pinchcock</td>
			<td>Nanjing Kangluoda Experimental Technology</td>
			<td>&#160;TB-624973311743</td>
			<td>36 mm &#215; 11 mm&#160; &#215; 55 mm</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3123000012</td>
			<td>10&#8211;100 &#181;L</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCL)</td>
			<td>Solarbio Life Science</td>
			<td>P9921</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Rubber tube</td>
			<td>Nanjing Kangluoda Experimental Technology</td>
			<td>TB-624923457094</td>
			<td>7 mm &#215; 5 mm</td>
		</tr>
		<tr>
			<td>Sterilizer</td>
			<td>BinJiang&#160;</td>
			<td>https://detail.1688.com/offer/644766597053.html?_t=1730892390541&#38;spm= 
			a2615.7691456.co_0_0_wangpu 
			_score_0_0_0_0_1_0_0000_0.0</td>
			<td>75L</td>
		</tr>
		<tr>
			<td>Tissue paper&#160;</td>
			<td>Heart to heart imprint</td>
			<td>M3</td>
			<td>22.5 cm &#215; 21.5 cm</td>
		</tr>
	</tbody>
</table>

inducing the entry of third stage dispersal juveniles of bursaphelenchus xylophilus into cryptobiosis through osmotic regulation

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal third-stage juveniles (pre-dauer juveniles, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" />) of Bursaphelenchus xylophilus can enter cryptobiosis through dehydration and revive upon rehydration when environmental conditions improve. Osmotic regulation is crucial for their survival in this process. In this study, specimens of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus were collected from dead Pinus massoniana due to pine wilt disease in Ningbo, Zhejiang Province, China. Following immersion in 8% potassium chloride (KCl) solution, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus entered a cryptobiotic state after gradual dehydration due to increased external osmotic pressure by natural water evaporation. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus could resist low-temperature stress at -20 &#176;C. <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus could revive upon rehydration, with a survival rate of 92.1%. This process can regulate the entry of <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus into cryptobiosis, enabling them to resist extreme environments. This method described in our study is simple and reliable, providing technical support for studying the stress resistance mechanisms of B. xylophilus.

In the 8% KCl solution, as the water evaporated, <img alt="Equation 1" src="/files/ftp_upload/67350/67350eq01.jpg" style="margin: auto;" /> B. xylophilus initially exhibited a noticeable &#34;curling&#34; while remaining motile (Figure 1A). Once the water evaporated completely, the nematodes ceased movement and curled, with depressed surfaces and accumulated lipid droplets (Figure 1B), indicating their entry into the cryptobiotic state. Upon the additio...

Inducing the Entry of Third Stage Dispersal Juveniles of Bursaphelenchus xylophilus into Cryptobiosis Through Osmotic Regulation

The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, J&#8546;) of Bursaphelenchus xylophilus into cryptobiosis through osmotic regulation of potassium chloride (KCl) ions. This method can provide technical support for research on the stress resistance mechanisms of B. xylophilus.

Cryptobiosis is a state where organisms lose nearly all their internal water and enter anhydrobiosis under extreme environmental stress. The dispersal ...

inducing-entry-third-stage-dispersal-juveniles-bursaphelenchus

Research

JoVE Journal

Biology

246 Views.  Chinese Academy of Forestry. The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, JⅢ) of Bursaphelenchus xylophilus into cryptobiosis through osmotic regulation of potassium chloride (KCl) ions. This method can provide technical support for research on the stress resistance mechanisms of B. xylophilus.

Video: Inducing the Entry of Third Stage Dispersal Juveniles of  Bursaphelenchus xylophilus  into Cryptobiosis Through Osmotic Regulation

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...