Figures 1 and 2 were previously published in Free Radical Biology &#38; Medicine (Queliconi&#160;et al.5) and have copyright held by Elsevier. This work was supported by the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP), the Instituto Nacional de Ci&#234;ncia e Tecnologia de Processos Redox em Biomedicina, the N&#250;cleo de Apoio &#224; Pesquisa de Processos Redox em Biomedicina, USPHS NS064945 (K.N.), and USPHS GM087483 (K.N.). B.B.Q. is a doctoral student supported by a FAPESP fellowship.

1. C. elegans&#160;Growth
<ol>
	<li>Prepare 35&#160;mm Nematode Growth Media (NGM) agar plates seeded with OP50 bacteria, as per standard cultivation methods1.</li>
	<li>Synchronize C. elegans by placing 6 gravid adults onto a seeded NGM plate. Remove the adults after ~100 eggs have been laid (~3 hr).</li>
	<li>Incubate the plates for 3 days at 20 &#176;C, at which point the worms will have developed to young adult stage and are optimal for this experiment. Alternative protocols to synchronize C. elegans are described elsewhere10.</li>
</ol>
2. Materials for AS
<ol>
	<li>M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4) should be purged of oxygen by equilibrating with N2 (argon can also be used) during 30 min prior to the experiment and kept surrounded by ice.</li>
	<li>Make a small hole (3&#160;mm wide) in the lid of 1.5&#160;ml&#160;microcentrifuge tubes in which the worms will be placed during AS. The hole allows for gas exchange without keeping the lid open, as this can lead to excessive media evaporation.</li>
</ol>
3. Anoxia/Starvation
<ol>
	<li>Conduct all experiments at least in triplicate.</li>
	<li>Add 1 ml&#160;of RT, oxygenated M9 buffer to each 35&#160;mm plate containing young adult worms grown as described above. Be careful to avoid removing bacteria from the plate (add M9 to the edges where bacteria weren&#39;t seeded). After ~1 min the worms should be swimming in the M9.</li>
	<li>Coat a 1&#160;ml&#160;pipette tip with BSA by pipetting and expelling a sterile 1% BSA solution.</li>
	<li>Carefully incline the plate containing the worms in M9 buffer, remove the M9 from the same place where it was added, and place the suspension in the prepared microcentrifuge tubes. Let rest in ice until the worms have dropped to the bottom of the tubes (~1-2 min).</li>
	<li>Remove the maximum amount of M9 possible without disturbing the worms from the tube (leaving around 100 &#956;l), add 1 ml&#160;of the oxygen-purged and iced M9 and place the tube over ice until the worms settle to the bottom again. Repeat the last step 3x. In the last wash, remove the M9 until ~100 &#956;l&#160;remains in the tube.</li>
	<li>Place the tubes with the worms in deoxygenated M9 inside an anoxic/hypoxic chamber (see instructions below, Section&#160;3.6.1) or, alternatively, into a custom sealed container (see instructions below, Section 3.6.2).
	<ol>
		<li>If a commercial anoxic/hypoxic chamber is to be used, leave some purged M9 inside the chamber before starting step 3.5 and repeat step 3.5 once the worms are inside the chamber.</li>
		<li>If a custom chamber is to be used, a detailed description of how to create a low-cost apparatus is presented at the end of this protocol and has been described by another group previously11. Briefly:
		<ol>
			<li>Make two holes in a 250&#160;ml&#160;Tupperware-type container lid and add tube connectors to them. One will be used to inject the gas mixture while the other will be the gas exit into a water bath.</li>
			<li>Place the worms into the chamber and seal the chamber using the lid.</li>
			<li>Gas with a constant N2 flux (100 ml/min).</li>
			<li>Place the container inside a water bath at 26 &#176;C. Ensure that the exit tube is well immersed in water so no air returns and that there are no leaks in the system (apparent by bubbles arising from the chamber itself).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubate the worms at 26 &#176;C for 20 hr.</li>
</ol>
4. Simulated Reperfusion
<ol>
	<li>After 20 hr under AS conditions, remove the tubes from the incubation chamber into room air.</li>
	<li>Pipette C. elegans with a BSA-coated tip (as in step 3.3) onto an OP50 seeded 35&#160;mm NGM plate. Incubate the plates at 20 &#176;C for 24 hr. After this, the worms will be ready to be scored.</li>
</ol>
5. Identifying Dead Versus Live Worms
<ol>
	<li>Using a platinum pick, lightly touch the top of the head of the worm. Live worms will move backwards after being touched. If the worms are nonresponsive, score as dead. Occasionally, a worm will not move backwards, but may exhibit a slight side-to-side head movement. Although not technically dead, these worms are almost certain to expire within hours, and should be scored as dead. If one waits too long to score the worms, internal hatching of progeny can occur and these &#8220;bags-of-worms&#8221; can rupture, making detecting the carcasses very difficult.</li>
	<li>Remove both live and dead worms from the plate as they are counted to avoid duplicate counts. The live worms can be moved to a separate plate to perform behavioral assays. The amount of living worms relative to the total is represented as the % of surviving worms (Figure&#160;1A).</li>
</ol>
6. Touch Response Assay
<ol>
	<li>A detailed description of this protocol has been presented in Hart, Anne C., ed. Behavior (July 3, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.87.1, http://www.wormbook.org.
	<ol>
		<li>To score for touch response, identify worms that are moving and lightly touch the side of the worm&#39;s head (near the middle part of the pharynx) with an eyelash pick (Figure&#160;3).</li>
		<li>If the worm moves backward, score as responsive, if not, score as nonresponsive (Figure&#160;1B). Repeat this step 10-15x for each worm with 10 sec intervals. Probe at least 10 worms in each group to have accurate data. The same assay can be used to assess forward locomotory changes following light touch to the posterior body wall. Be sure not to touch the head or tail of the worm, since this stimulates a distinct behavior.</li>
		<li>Finally, incorporate genetic control strains [N2 Bristol wild-type for positive, mec (mechanosensory) mutant for negative -&#160;e.g. CB1338 mec-3 (e1338)IV] to calibrate the force with which to touch the worms. Too harsh of a stimulation will elicit a behavioral response in mec mutants, too little will not elicit an effect in wildtype N2 controls.</li>
	</ol>
	</li>
</ol>
7. Neuronal Modifications
<ol>
	<li>To visualize post AS neuronal modifications, use a strain which expresses GFP in the touch-responding mec neurons4,5. These neurons have long processes that run along the body wall and are easily scored for morphological abnormalities. In addition, the morphologic information can be integrated with behavioral measures of neuronal function, as described above. One strain that can be used for this purpose is TU2583, which is available from the C. elegans Genetics Center and contains the uIs25 integrated transgene expressing GFP from the mec-18 promoter, or alternatively TU2562, which contains an integrated mec-3::GFP fusion.</li>
	<li>Prepare an agarose pad.
	<ol>
		<li>Melt 2% agarose in water, bring to 1x M9 with a 10x stock, and maintain the solution at 70 &#176;C.</li>
		<li>Place a drop (~20 &#956;l) of this solution on a glass slide (25 mm x 75 mm) that has been placed between two other slides, each of which have a single piece of tape on their bottom side.</li>
		<li>Place a final glass slide perpendicular to the first on top of the 2% agarose solution, and let it cool at RT. This should form a pad that is the thickness of the tape.</li>
		<li>Remove the top slide and add 10 &#956;l&#160;of M9 containing 0.1% tetramisole, which will immobilize the worms.</li>
	</ol>
	</li>
	<li>Move some (~10) live worms onto the 2% agarose slide. Place a coverslip over them and visualize GFP using a fluorescence microscope under a 100X&#160;objective with the appropriate illumination.</li>
	<li>Damaged neurons will show a cytosolic membrane string of pearls pattern in the processes, comprised of multiple punctum (Figure&#160;2A), and/or abnormalities where the processes appear to be broken (Figure&#160;2B). Count punctum and abnormalities in both neurons for each worm, using a total of at least 10 worms.</li>
</ol>
8. Lab-made Hypoxic Chamber
<ol>
	<li>A Tupperware-style, sealable, airtight container can be retrofit as an anoxic chamber. Take care to select a container that is as small as feasible. A small container creates a hypoxic environment faster and is easier to fit in the water bath (Figure&#160;4).</li>
	<li>Make two holes on the lid (can be done with a screwdriver or by heating forceps and forcing into the lid) and insert a plastic or metal connector that fits the tubing (Figure&#160;5A). Use glue that can be placed under water, and also gets hard after drying, avoiding movement of the connector and hence reducing the possibility of leakage (Figure&#160;5B).</li>
	<li>The container should be placed into the water bath so that it is submerged completely (Figure&#160;4). To immerse the container, use a bottle weight or a diving weight.</li>
	<li>One of the tube connections will be used to introduce a gas mixture and the other tube will act as a pressure relief, hence it should be left under the water so that the container is sealed off from the external environment (Figure&#160;4).</li>
	<li>The air composition can be modulated by using custom made gas, mass flow controllers or any apparatus that mixes the desired gases. The gas flow should be controlled, as high flow can create excessive evaporation. For example, in a 250 ml&#160;container, 100 ml/min of gas will provide good space for the worms and a suitable flux.</li>
	<li>The length of the tubing should be kept to a minimum, as they can be permeable to gas, allowing the entrance of O2. It is important to avoid using silicone, Tygon tubes as these materials are permeable to gas exchange. We prefer polypropylene or nylon tubes. Glass or metal tubing maintain the gasses well, but are also harder to work with.</li>
</ol>

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Maine, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+gene+function+in+Caenorhabditis+elegans+using+RNA-mediated+interference.">Studying gene function in Caenorhabditis elegans using RNA-mediated interference.</a> Briefings Funct. Genom. Proteom. 7 (3), 184-194 (2008).</li><li>Vanden Hoek, ., L, T., Shao, Z., Li, C., Zak, R., Schumacker, P. T., Becker, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+injury+on+cardiac+myocytes+after+simulated+ischemia.">Reperfusion injury on cardiac myocytes after simulated ischemia.</a> Am. J. Physiol. 270 (4), 1334-1341 (1996).</li><li>Ba Scott, ., Avidan, M. S., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+hypoxic+death+in+C.+elegans+by+the+insulin/IGF+receptor+homolog+DAF-2.">Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2.</a> Science. 296 (5577), 2388-2391 (2002).</li><li>Queliconi, B. B., Marazzi, T. B. M., 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=Bicarbonate+modulates+oxidative+and+functional+damage+in+ischemia-reperfusion.">Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion.</a> Free Rad. Biol. Med. 55, 46-53 (2013).</li><li>Wojtovich, A. P., DiStefano, P., Sherman, T., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP-sensitive+potassium+channel+activity+and+hypoxic+preconditioning+are+independent+of+an+inwardly+rectifying+potassium+channel+subunit+in+Caenorhabditis+elegans.">Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.</a> FEBS Lett. 586 (4), 428-434 (2012).</li><li>Dasgupta, N., Patel, A. M., Scott, B. A., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic+Preconditioning+Requires+the+Apoptosis+Protein+CED-4+in+C.+elegans.">Hypoxic Preconditioning Requires the Apoptosis Protein CED-4 in C. elegans.</a> Curr. Biol. 17 (22), 1954-1959 (2007).</li><li>Jia, B., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+anesthetic+preconditioning+present+in+the+invertebrate+Caenorhabditis+elegans.">Volatile anesthetic preconditioning present in the invertebrate Caenorhabditis elegans.</a> Anesthesiology. 108 (3), 426-433 (2008).</li><li>Wojtovich, A. P., Sherman, T. A., Nadtochiy, S. M., Urciuoli, W. R., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLO-2+is+cytoprotective+and+contributes+to+mitochondrial+potassium+transport.">SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.</a> PloS One. 6 (12), e28287 (2011).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Fawcett, E. M., Horsman, J. W., Miller, 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=Creating+defined+gaseous+environments+to+study+the+effects+of+hypoxia+on+C.+elegans.">Creating defined gaseous environments to study the effects of hypoxia on C. elegans.</a> J. Vis. Exp. (65), e4088 (2012).</li><li>Butler, J. A., Mishur, R. J., Bokov, A. F., Hakala, K. W., Weintraub, S. T., Rea, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+anaerobic+response+of+C.+elegans+using+GC-MS.">Profiling the anaerobic response of C. elegans using GC-MS.</a> PLoS One. 7 (9), 10-1371 (2012).</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 (5), 1473-1483 (2002).</li><li>Jiang, H., Guo, R., Powell-Coffman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+hif-1+gene+encodes+a+bHLH-PAS+protein+that+is+required+for+adaptation+to+hypoxia.">The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7916-7921 (2001).</li><li>Twumasi-Boateng, K., Wang, T. W., 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=An+age-dependent+reversal+in+the+protective+capacities+of+JNK+signaling+shortens+Caenorhabditis+elegans+lifespan.">An age-dependent reversal in the protective capacities of JNK signaling shortens Caenorhabditis elegans lifespan.</a> Aging Cell. 11 (4), 659-667 (2012).</li><li>Wang, Y., Chen, J., Wei, G., He, H., Zhu, X., Xiao, T., Yuan, J., Dong, B., He, S. S. k. o. g. e. r. b. &. #. 2. 4. 8. ;. G., Chen, 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+Caenorhabditis+elegans+intermediate-size+transcriptome+shows+high+degree+of+stage-specific+expression.">The Caenorhabditis elegans intermediate-size transcriptome shows high degree of stage-specific expression.</a> Nucleic Acids Res. 39 (12), 5203-5214 (2011).</li><li>Perry, C. N., Huang, C., Liu, W., Magee, N., Carreira, R. S., Gottlieb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+mitochondrial+electron+transfer+enzyme,+Ndi1,+in+myocardial+reperfusion+injury.">Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.</a> PloS One. 6 (2), e16288 (2011).</li><li>Chai, Y., Li, W., Feng, G., Yang, Y., Wang, X., Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cellular+Dynamics+During+Caenorhabditis+elegans+Postembryonic+Development.">Live Imaging of Cellular Dynamics During Caenorhabditis elegans Postembryonic Development.</a> Nature Protoc. 7 (12), 2090-2102 (2012).</li><li>Nehrke, 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+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J. Biol. Chem. 278 (45), 44657-44666 (1074).</li></ol>

The authors have nothing to disclose.

AS has been widely used in C. elegans to model IR injury. Some key points should be highlighted for this protocol: C. elegans are resistant to a wide array of injuries, justifying the need for 3 concomitant insults (heat, starvation and anoxia) to achieve death using this system. Anoxia alone does not kill the worms in this window of time14. Furthermore, temperature increase is an additional stress, so it is important to monitor closely. Strictly speaking, starvation does not contribute significantly to the degree of mortality observed7, per se, but it appears to reduce variability among experimental replicates. Given that there can be significant variability from day-to-day, it is extremely important to compare samples run directly in parallel, and to repeat experiments over multiple days. In general, outcomes are measured for three separate plates of 50-100 worms/experimental condition and these are then averaged and considered as a single experimental replicate. Generally, between seven and nine replicates appear to be sufficient to achieve or rule out statistical significance.
The developmental stage of the worms used in the experiment also needs to be carefully monitored as the susceptibility of different stages to AS damage varies significantly15,16. The use of young adults is standard, and larval stage (L3 and L4) worms appear to be more resistant to the damaging effects of AS (unpublished data).
This protocol presents two ways to perform the experiments, one using a lab-made apparatus (using Tupperware-type containers and gas input11) and other using a commercial hypoxic chamber4,6. Anoxia can be achieved by other means that consume the oxygen, as described elsewhere13,17. The use of alternate techniques to create a hypoxic environment may change the AS incubation time necessary to create the desired amount of death. Targeting ~20% survival is an ideal starting point for studying protective interventions, while 80% is similarly ideal for interventions that exacerbate the detrimental effects of AS. Another important caveat is the time at which the observer scores dead/alive worms. If the time for analysis is extended beyond 24 hr, the data may be misleading since dead worms become increasingly difficult to identify. This may be due to worm carcasses becoming relatively transparent over time, but also to the fact that fertilized embryos can develop into progeny post-mortem inside of the carcasses and disrupt them as they emerge.
The analysis of neuronal morphology can be modified to look at protein expression patterns18, nuclear fragmentation4 and other parameters19 by substituting a worm strain that expresses the appropriate genetically encoded marker. One final caveat is that the visualization of the neuronal processes should be done less than 30 min after placing the worms on the slide. Animals kept under anesthesia on slides for longer periods can exhibit AS-independent damage. Adjust the amount of animals per slide according to the time needed to track and analyze them.

C. elegans is a nematode that has been widely adopted as a multicellular eukaryotic model organism since its introduction by Brenner1. It is a cheap, simple, and versatile model, which allows easy links between genetic alterations and phenotypic changes2.
Ischemia is characterized by a lack of nutrients and oxygen supply to a tissue, followed by reperfusion, when a burst of reactive oxygen species is produced3 and most of the damage occurs. In 2002, a model of ischemia/reperfusion (IR) in C. elegans was developed4 involving submitting the whole worm to anoxia, nutrient deprivation and heat stress for approximately 20 hr&#160;followed by 24 hr&#160;under normal conditions. Although this model is technically an anoxia-starvation (AS) condition, cell death occurs through mechanisms that are conserved in mammals, including damage induced by oxidants during reperfusion5. Furthermore, similar to mammalian IR, damage induced by AS in C. elegans can be prevented by ischemic preconditioning6,7 or anesthetic preconditioning8,9.
The protocols below demonstrate how to mimic IR in C. elegans using the AS model, how to score morphological and behavioral abnormalities that result from AS, and how to adapt the protocol in a way that allows the experiment to be conducted with a lower initial investment using a custom-made, easily-constructed chamber alternative.

<table><tbody><tr><td>N2&amp;nbsp; strain</td><td>CGC</td><td></td><td>Wild type strain.&lt;p&gt; http://www.cbs.umn.edu/CGC/&lt;/p&gt;</td></tr><tr><td>TU2583 uIs25 (P&lt;em&gt;mec-18&lt;/em&gt;::GFP)</td><td>CGC</td><td>TU2583</td><td>integrated fluorescent transgene used to label touch neurons.&lt;p&gt;
http://www.cbs.umn.edu/CGC/&lt;/p&gt;</td></tr><tr><td>CB1338 &lt;em&gt;mec-3&lt;/em&gt; (&lt;em&gt;e1338&lt;/em&gt;)IV</td><td>CGC</td><td>CB1338</td><td>canonical mec-3 mutant that is touch insensitive.&lt;p&gt;
http://www.cbs.umn.edu/CGC/&lt;/p&gt;</td></tr><tr><td>Microcentrifuge Tube</td><td>Eppendorf</td><td>0030 120.086</td><td></td></tr><tr><td>Nikon Eclipse TE2000-U Microscope&amp;nbsp;</td><td>Nikon USA</td><td>TE2000-U</td><td></td></tr><tr><td>Low Temperature Incubator</td><td>Sheldon Manufacturing Inc.</td><td>Model 2005</td><td></td></tr><tr><td>Eyelash Pick</td><td></td><td></td><td>An eyelash pick can be prepared by attaching&amp;nbsp; an eyelash onto a wooden toothpick, then attaching the toothpick in a glass Pasteur pipette (&lt;strong&gt;Figure 3&lt;/strong&gt;).</td></tr><tr><td>Hypoxic Chamber</td><td>Coy</td><td>8307030</td><td>Hypoxic Glove box equipped with paladium catalyst and CO&lt;sub&gt;2&lt;/sub&gt; controller.</td></tr></tbody></table>

an anoxia-starvation model for ischemia/reperfusion in c. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

Subjecting C. elegans to 20 hr&#160;of AS at 26 &#176;C in a custom lab-made incubation chamber as described (Section 3.6.2) resulted in significant mortality (Figure 1A)5. Subsequent fluorescent imaging of punctum and breaks in the GFP labeled neuronal processes of survivors confirmed the presence of morphological abnormalities (Figure&#160;2). The survivors also responded poorly to light body wall touch (Figure&#160;1B). This model has been used by multiple groups to study how genetic predisposition, pharmaceutical intervention, and metabolic plasticity affects AS dependent outcomes4-6,8,9,12,13.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig1highres.jpg" src="/files/ftp_upload/51231/51231fig1.jpg" /> 
Figure 1. C. elegans survival and touch response after AS.&#160;A)&#160;Percentage of worms that exhibit 24 hr post-AS survival. The N2 strain is the wild type genetic background, and KWN85 contains an integrated transgene that labels mec neurons with GFP.&#160;B)&#160;Response to touch stimuli of living C. elegans (N2 Strain) after AS. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51231/51231fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig2highres.jpg" src="/files/ftp_upload/51231/51231fig2.jpg" /> 
Figure 2. Touch neuron modifications after AS. Touch neuron (PLML and PLMR) abnormalities such as tortuous processes and breaks (A) or the accumulation of GFP aggregates in the processes (B) were monitored in surviving anesthetized C. elegans after AS.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig3highres.jpg" src="/files/ftp_upload/51231/51231fig3.jpg" /> 
Figure 3. Eyelash pick. An assembled eyelash pick. In the inset, a detail of the eyelash glued to the toothpick wood.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig4highres.jpg" src="/files/ftp_upload/51231/51231fig4.jpg" /> 
Figure 4. Lab-made hypoxic chamber inside the water bath. A closed container ready to start the experiment. Arrows indicate the gas entrance and exit and the bottle weight used to keep it from floating.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig5highres.jpg" src="/files/ftp_upload/51231/51231fig5.jpg" /> 
Figure 5. Details of the internal and external side of the container lid. A) External side of the container lid, indicating the tube and its connector attached to the previously made hole. B)&#160;External side of the container lid, indicating the tube connector attached to the previously made hole.

Watch this Scientific Journal Video about An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans at JoVE.com

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

an-anoxia-starvation-model-for-ischemiareperfusion-in-c-elegans

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.8K Views.  Universidade de São Paulo. A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Video: An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Developmental Biology

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

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.

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

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

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.

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.

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Neuroscience

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

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

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans