We thank members of the Miller lab for discussion and critical reading of the manuscript. This work was supported by a new investigator award from the Nathan Shock Center of Excellence in the Basic Biology of Aging to DLM and the National Institutes of Health award R00 AG030550 to DLM.

1. Construction of Environmental Chambers
<ol>
	<li>Select the smallest reasonable volume of chamber required for the scope of your project. Chamber must be made of gas (O2) impermeable material. Pyrex crystallization dishes, Anaeropack boxes, or large cast-acrylic boxes (Ellard Instrumentation), can be used. We have found that 9 50 mm plates can fit in a 100 x 50 Kimex crystallization dish. Glass plates can be used as lids for Pyrex crystallization dishes.</li>
	<li>Drill a hole in the selected chamber and ...

<ol>
	<li>Danovaro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+metazoa+living+in+permanently+anoxic+conditions.">The first metazoa living in permanently anoxic conditions.</a> BMC Biology. 8, 30 (2010).</li><li>Birner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Hypoxia-inducible+Factor+1+alpha+Is+a+Marker+for+an+Unfavorable+Prognosis+in+Early-Stage+Invasive+Cervical+Cancer.">Overexpression of Hypoxia-inducible Factor 1 alpha Is a Marker for an Unfavorable Prognosis in Early-Stage Invasive Cervical Cancer.</a> Cancer Research. 60, 4693-4696 (2000).</li><li>Harris, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+-+a+key+regulatory+factor+in+tumour+growth.">Hypoxia - a key regulatory factor in tumour growth.</a> Nat. Rev. Cancer. 2, 38-47 (2002).</li><li>Ramirez-Bergeron, 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=Hypoxia+affects+mesoderm+and+enhances+hemangioblast+specification+during+early+development.">Hypoxia affects mesoderm and enhances hemangioblast specification during early development.</a> Development. 131, 4623-4634 (2004).</li><li>Staff, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheel-well+Stowaways+Risk+Lethal+Levels+of+Hypoxia+and+Hypothermia.">Wheel-well Stowaways Risk Lethal Levels of Hypoxia and Hypothermia.</a> Human Factors and Aviation. 44, 1-5 (1997).</li><li>Shen, C., 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=Genetic+Analysis+of+Hypoxia+Signaling+and+Response+in+C.+elegans.">Genetic Analysis of Hypoxia Signaling and Response in C. elegans.</a> Annals of the New York Academy of Sciences. 995, 191-199 (2003).</li><li>Shen, C., Nettleton, D., Jiang, M., Kim, S. K., 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=Roles+of+the+HIF-1+Hypoxia-inducible+Factor+during+Hypoxia+Response+in+Caenorhabditis+elegans.">Roles of the HIF-1 Hypoxia-inducible Factor during Hypoxia Response in Caenorhabditis elegans.</a> Journal of Biological Chemistry. 280, 20580-20588 (2005).</li><li>Wang, G. L., Jiang, B. H., Rue, E. A., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1+is+a+basic-helix-loop-helix-PAS+heterodimer+regulated+by+cellular+O2+tension.">Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.</a> Proceedings of the National Academy of Sciences. 92, 5510-5514 (1995).</li><li>Epstein, A. C. R. <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+EGL-9+and+Mammalian+Homologs+Define+a+Family+of+Dioxygenases+that+Regulate+HIF+by+Prolyl+Hydroxylation.">C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation.</a> Cell. 107, 43-54 (2001).</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>Gray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensation+and+social+feeding+mediated+by+a+C.+elegans+guanylate+cyclase+homologue.">Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue.</a> Nature. 430, 317-322 (2004).</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> Current Biology. 19, 1233-1237 (2009).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C. M., 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> Molecular Biology of the Cell. 13, 1473-1483 (2002).</li><li>Hu, C. -. J., Wang, L. -. Y., Chodosh, L. A., Keith, B., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Roles+of+Hypoxia-Inducible+Factor+1{alpha}+(HIF-1{alpha})+and+HIF-2{alpha}+in+Hypoxic+Gene+Regulation.">Differential Roles of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}) and HIF-2{alpha} in Hypoxic Gene Regulation.</a> Molecular and Cellular Biology. 23, 9361-9374 (2003).</li><li>Treinin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+is+required+for+heat+acclimation+in+the+nematode+Caenorhabditis+elegans.">HIF-1 is required for heat acclimation in the nematode Caenorhabditis elegans.</a> Physiological Genomics. 14, 17-24 (2003).</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=Hydrogen+sulfide+increases+thermotolerance+and+lifespan+in+Caenorhabditis+elegans.">Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans.</a> PNAS. 104, 20618-20622 (2007).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Massie, M. R., Lapoczka, E. M., Boggs, K. D., Stine, K. E., White, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+the+metabolic+inhibitor+sodium+azide+induces+stress+protein+expression+and+thermotolerance+in+the+nematode+Caenorhabditis+elegans.">Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans.</a> Cell Stress Chaperones. 8, 1-7 (2003).</li><li>Salceda, S., Caro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+Factor+1&#945;+(HIF-1&#945;)+Protein+Is+Rapidly+Degraded+by+the+Ubiquitin-Proteasome+System+under+Normoxic+Conditions.">Hypoxia-inducible Factor 1&#945; (HIF-1&#945;) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions.</a> Journal of Biological Chemistry. 272, 22642-22647 (1997).</li><li>Theilacker, J. C., White, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+Gases+in+Air+and+Its+Affect+on+Oxygen+Deficiency+Hazard+Abatement.">Diffusion of Gases in Air and Its Affect on Oxygen Deficiency Hazard Abatement.</a> AIP Conference Proceedings. 823, 305-312 (2006).</li><li>Chua, B., Kao, R. L., Rannels, D. E., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+degradation+by+anoxia+and+ischemia+in+perfused+rat+hearts.">Inhibition of protein degradation by anoxia and ischemia in perfused rat hearts.</a> Journal of Biological Chemistry. 254, 6617-6623 (1979).</li><li>Probst, G., Riedinger, H., Martin, P., Engelcke, M., Probst, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Control+of+DNA+Replication+in+Response+to+Hypoxia+and+to+Inhibited+Protein+Synthesis+in+CCRF-CEM+and+HeLa+Cells.">Fast Control of DNA Replication in Response to Hypoxia and to Inhibited Protein Synthesis in CCRF-CEM and HeLa Cells.</a> Biological Chemistry. 380, 1371-1382 (1999).</li><li>Semenza, G. L., Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oxygen Sensing. 381, (2004).</li><li>Chan, K., 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=Anoxia-Induced+Suspended+Animation+in+Budding+Yeast+as+an+Experimental+Paradigm+for+Studying+Oxygen-Regulated+Gene+Expression.">Anoxia-Induced Suspended Animation in Budding Yeast as an Experimental Paradigm for Studying Oxygen-Regulated Gene Expression.</a> Eukaryotic Cell. 7, 1795-1808 (2008).</li><li>Nystul, T. G., 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=Carbon+monoxide-induced+suspended+animation+protects+against+hypoxic+damage+in+Caenorhabditis+elegans.">Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans.</a> PNAS. 101, 9133-9136 (2004).</li></ol>

This method presents a strategy for constructing a hypoxic environment that allows for environments with precise concentrations of oxygen to be maintained in the laboratory. These chambers provide a simple method for exposing organisms to specific low concentrations of O2 and monitoring the molecular and physiological outputs. The environmental chamber described is assembled by the lab instead of commercially purchased and can thus be modified to fit the needs of the experiment.
One...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Tubing (FEP and PTFE)</td>
			<td>Cole Parmer 
			Tygon</td>
			<td>YO-95821-00 (1/8" FEP) 
			06605-27 (1/16 x 1/8" PTFE)' R-3603</td>
		</tr>
		<tr>
			<td>Compression fittings</td>
			<td>Seattle Fluid Systems</td>
			<td>06363-58 (M. coupler 1/16") 
			06363-62 (F. coupler 1/16") 
			06363-60 (M. coupler 1/8") 
			06363-61 (F. coupler 1/8")</td>
		</tr>
		<tr>
			<td>Flow tube</td>
			<td>Aalborg</td>
			<td>PMR3-010073 (3 output) 
			PMR1-013520 (1 output)</td>
		</tr>
		<tr>
			<td>Mass flow controller</td>
			<td>Sierra Instruments</td>
			<td>810S-L-DR-2-OV1-SK1-V1-S1 (Mass Trak) 
			C100L-DD-2-OV1-SV1-PV2-V1-SO-C10 (Smart Trak 2)</td>
		</tr>
		<tr>
			<td>Compressed gas tank</td>
			<td>AirGas</td>
			<td>Made to order</td>
		</tr>
		<tr>
			<td>Plastic male Luer to hose barb fittings</td>
			<td>Cole Parmer</td>
			<td>45505-41 (500 series 1/16")</td>
		</tr>
		<tr>
			<td>Cast acrylic boxes</td>
			<td>Ellard Instrumentation</td>
			<td>Made to order</td>
		</tr>
		<tr>
			<td>Pipe fittings (Brass or stainless steel)</td>
			<td>Seattle Fluid Systems</td>
			<td>B-402-1 (1/4" nut) 
			B-200-3 (1/8" union tee) 
			B-400-set (1/4" ferrules) 
			B-QM2-B1-200 (QM Body QC) 
			B-200-1-2 (1/8 x 1/8" male conn)</td>
		</tr>
		<tr>
			<td>Dow Corning Vacuum Grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
		</tr>
		<tr>
			<td>AnaeroPack box</td>
			<td>Misubishi Gas Chemical Company</td>
			<td>R684004 (0.4 liter) 
			R685025 (2.5 liter) 
			R685070 (7.0 liter)</td>
		</tr>
		<tr>
			<td>Pyrex gas wash bottle</td>
			<td>Sigma-Aldrich</td>
			<td>CLS31770500C (500 mL) 
			CLS31770250C (250 mL) 
			CLS31770125C (125 mL)</td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Sigma-Aldrich</td>
			<td>P0500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-horseradish peroxidase</td>
			<td>Southern Biotechnology Associates</td>
			<td>1032-05</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminsecent Substrate</td>
			<td>Pierce Chemical</td>
			<td>34077</td>
		</tr>
		<tr>
			<td>100 x 50 glass crystallization dishes</td>
			<td>Kimax Kimble</td>
			<td>23000</td>
		</tr>
	</tbody>
</table>

creating defined gaseous environments to study the effects of hypoxia on c. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Watch this Scientific Journal Video about Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans at JoVE.com

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

creating-defined-gaseous-environments-to-study-effects-hypoxia-on-c

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12.0K visualizações.  University of Washington. Este artigo detalha como usar contínuo fluxo de câmaras de hipóxia para gerar atmosferas com concentrações definidas de O 2 Para entender as respostas biológicas à diminuição da O 2. Este sistema é fácil de configurar e manter, e flexível o suficiente para atender uma ampla gama de O 2 Concentrações e sistemas modelo