We would like to convey our appreciation for the input and comments from members of the Padilla Lab. Nematode strains in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). We acknowledge and thank Dr. Lon Turnbull for technical assistance with confocal microscopy. This work has been supported by a grant from the National Science Foundation (NSF-IOS, CAREER) to P.A.P.

1. Sample Preparation
<ol>
	<li>Produce or obtain appropriate transgenic C. elegans strain of interest using transgenic methodologies or from Caenorhabditis Genetics Stock Center (CGC) or colleague. In this case we are using strain TH32 (pie-1::tbg-1::GFP; pie-1::GFP::H2B)5 to visualize chromosomes and centrosomes as markers for cell division.</li>
	<li>Generate a synchronized population by using one of three methods: 1) disintegrate gravid adults in hypochlorite solution and us...

<ol>
	<li>Powell-Coffman, 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=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+stress+in+Caenorhabditis+elegans.">Longevity and stress in Caenorhabditis elegans.</a> Aging (Albany NY). 3, 733-753 (2011).</li><li>Schmidt, D. J., Rose, D. J., Saxton, W. M., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cytoplasmic+dynein+heavy+chain+in+Caenorhabditis+elegans+with+fast-acting+temperature-sensitive+mutations.">Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations.</a> Mol. Biol. Cell. 16, 1200-1212 (2005).</li><li>Sulston, J., Hodgkin, J., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+sub-nuclear+changes+in+Caenorhabditis+elegans+embryos+exposed+to+brief,+intermediate+and+long-term+anoxia+to+analyze+anoxia-induced+cell+cycle+arrest.">Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest.</a> BMC Cell Biol. 6, 1471-2121 (2005).</li><li>Padilla, P. A., Goy, J. M., Hajeri, P. A., Padilla, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anoxia. , (2012).</li><li>Hajeri, V. A., Little, B. A., Ladage, M. L., Padilla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NPP-16/Nup50+function+and+CDK-1+inactivation+are+associated+with+anoxia-induced+prophase+arrest+in+Caenorhabditis+elegans.">NPP-16/Nup50 function and CDK-1 inactivation are associated with anoxia-induced prophase arrest in Caenorhabditis elegans.</a> Mol. Biol. Cell. 21, 712-724 (2010).</li><li>Nystul, T. G., Goldmark, J. P., Padilla, P. A., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation+in+C.+elegans+requires+the+spindle+checkpoint.">Suspended animation in C. elegans requires the spindle checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Margalit, A., Vlcek, S., Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+making+of+the+nuclear+envelope.">Breaking and making of the nuclear envelope.</a> J. Cell Biochem. 95, 454-465 (2005).</li><li>Maddox, A. S., Maddox, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cellular+processes+in+Caenorhabditis+elegans.">High-resolution imaging of cellular processes in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 1-34 (2012).</li><li>Miller, D. L., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+are+protected+from+lethal+hypoxia+by+an+embryonic+diapause.">C. elegans are protected from lethal hypoxia by an embryonic diapause.</a> Curr. Biol. 19, 1233-1237 (2009).</li></ol>

No conflicts of interest declared.

Oxygen Deprivation and Suspended Animation
Although exposure to severe oxygen deprivation can be fatal for some organisms, some organisms are able to survive exposure to anoxia. In the case of C. elegans, survival of anoxia exposure depends on developmental stage and response to anoxia is entry into a reversible state of suspended animation in which a number of observable biological processes are arrested. Cell division, development, movement, eating and reproductive processes cease unt...

<table border="1">
	<tbody>
		<tr>
			<td>Reagents/Equipment</td>
			<td></td>
			<td></td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Hypochlorite solution</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.7 g KOH, 12 ml 5% NaOCl, bring to 50 ml with ddH20</td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3 g KH2PO4, 11 g Na2HPO4, 5 g NaCl, 1 ml (1 M) MgSO4 per 1 L ddH2O</td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-343</td>
			<td>3"x1"x1.0 mm</td>
		</tr>
		<tr>
			<td>Round micro coverglass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72223-01</td>
			<td>25 mm Diameter</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma</td>
			<td>H8898-100ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.5% tricaine, 0.05% tetramisole</td>
		</tr>
		<tr>
			<td>Leiden Closed Perfusion Microincubator</td>
			<td>Harvard Apparatus</td>
			<td>650041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UHP Nitrogen</td>
			<td>Calgaz (Air Liquide)</td>
			<td>&nbsp;</td>
			<td>&gt;99.9990% N2 &lt;2 ppm O2</td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td>VWR</td>
			<td>89068-468</td>
			<td>0.062" ID x 0.125" OD</td>
		</tr>
		<tr>
			<td>Spinning Disk Confocal Microscope</td>
			<td>McBain Systems</td>
			<td>&nbsp;</td>
			<td>Zeiss inverted optical microscope, epifluorescence illumination system, CSU-10 Yokogawa confocal scanner, Hamamatsu electron multiplier CCD camera.</td>
		</tr>
	</tbody>
</table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

C. elegans embryos exposed to severe oxygen deprivation (anoxia) are able to survive by arresting biological processes including development and cell division7. The anoxia-induced arrest of cell division can be monitored, at a sub-cellular level, by using a microincubation chamber in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo (Figure 1). Cellular structures of int...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

use-time-lapse-microscopy-to-visualize-anoxia-induced-suspended

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Biology

11.5K visualizações.  University of North Texas. Descrito aqui é um In vivo A técnica de sub-imagem estruturas celulares em animais expostos a anoxia usando um fluxo de gás através da câmara microincubation em conjunto com um microscópio confocal de disco giratório. Este método é simples e suficientemente flexível para satisfazer uma variedade de parâmetros experimentais e sistemas modelo.