Name: measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates
Uploaded: 2015-05-09T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

We acknowledge the Caenorhabditis Genetics Center for C. elegans strains. Funding support was provided by the Terry Fox Research Institute. We also acknowledge support granted to E.P. from the Rolande and Marcel Gosselin Graduate Studentship and the CIHR/FRSQ training grant in cancer research FRN53888 of the McGill Integrated Cancer Research Training Program.

1. Preparation of Reagents
<ol>
	<li>Preparation of media for C. elegans growth (in this case, wild-type animals and flcn-1(ok975) mutant animals).
	<ol>
		<li>Prepare Modified Youngren&#39;s Only Bacto-peptone (MYOB) dry mix containing 5.5 g of Tris HCl, 2.4 g of Tris base, 31 g of Bactopetone, 20 g of NaCl and 0.08 g of cholesterol. Mix well with shaking. 
		NOTE: This mix is sufficient to prepare 10 L of MYOB medium. 
		NOTE: Normal growth medium (NGM) plates could be used instead of MYOB plates27. However, the same type of plates should be used for valid comparisons between strains and between independent repeats. In this protocol, we only used MYOB plates.</li>
		<li>Prepare 1 L of MYOB medium by adding 6 g of MYOB dry mix, 17 g of agar and make up to 1 L with H2O and autoclave for 45 min at 122 &#176;C. Pour plates and let dry at RT for 2 days.</li>
		<li>Using sterile technique, inoculate 100 ml of culture of LB broth medium with E. coli OP50 bacteria and grow O/N&#160;at 37 &#176;C.</li>
		<li>Seed MYOB plates with E. coli OP50 bacteria. Allow it to grow at RT for 48 hr.</li>
	</ol>
	</li>
	<li>Preparation for gene downregulation using RNAi
	<ol>
		<li>Prepare MYOB RNAi-feeding plates. Proceed as in steps 1.1.1 and 1.1.2. After autoclaving, allow the medium to cool down to about 55 &#176;C and add 1 ml of 1 M isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and 1 ml of 100 mM ampicillin.</li>
		<li>Streak E. coli HT115 bacteria transformed with a plasmid, which expresses either control EV or flcn-1 specific RNAi on Ampicillin (50 &#181;g/ml)/tetracyclin (15 &#181;g/ml) agar plates. Grow culture O/N at 37 &#176;C.</li>
		<li>Pick colonies and inoculate E. coli HT115 bacteria transformed with a plasmid which expresses either control EV or flcn-1 specific RNAi and grow culture in LB/Ampicillin (50 &#181;g/ml) medium and O/N at 37 &#176;C.</li>
		<li>Seed plates with E. coli HT115 EV bacteria or HT115 flcn-1 RNAi bacteria. 
		NOTE: Keep plates 48 hr at RT&#160;before usage. If RNAi knockdown by feeding is in effective, use other methods of delivery of the ds-RNA 28.</li>
	</ol>
	</li>
	<li>Inducing oxidative stress using paraquat (PQ):
	<ol>
		<li>Prepare M9 buffer by mixing 3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl and 0.25 g of MgSO4&#183;7 H2O. Bring up to 1 L with distilled water. Autoclave solution for 45 min at 122 &#176;C and store bottles at RT.</li>
		<li>On the day of the assay, prepare the M9/PQ-containing solution. For this protocol, use 100 mM PQ (0.1028 g of PQ dissolved in 4 ml of M9 buffer fills an entire 96 well plates with 40 &#181;l of M9/PQ per well). 
		CAUTION: PQ is a hazardous and highly toxic compound. Handle according to appropriate guidelines. 
		NOTE: When first running a new strain or a new condition, it is recommended to assay survival in increasing concentrations of PQ to determine the best concentration to use in order to kill the animals within 7-10 hr.</li>
	</ol>
	</li>
</ol>
2. Preparation of Age-synchronized L4 Population
<ol>
	<li>Transfer 20 gravid adult hermaphrodites to a fresh MYOB plate seeded with E. coli OP50 bacteria. Prepare several plates based on the number of worms needed during the assay.</li>
	<li>Allow them to lay eggs for 6 hr and then using a platinum wire worm pick remove the mothers. Check the plates under the microscope to assess the number of eggs laid.</li>
	<li>Allow the eggs to hatch and grow at 20 &#176;C for 48 hr. At this time, the animals are in a late L4/young adult stage. Pick same stage animals and transfer to the PQ solution-containing wells. 
	NOTE: Since 48 hr incubation only applies to mutants that grow similarly to wild-type animals, it is important to monitor developmental rate of newly tested mutants to ensure it conforms to the wild-type development rate, otherwise, other timelines should be used.</li>
	<li>Alternatively, use synchronization techniques. Generate synchronized population of L1 larvae worms using hypochlorite solution (25 ml water, 125 ml of 5.25% sodium hypochlorite, 50 ml of 4 M NaOH; wrap bottle with aluminum foil to isolate from light). However, for this assay, it is not recommended, since intense bleaching might affect the behavior of the animals during the assay. 
	CAUTION: Handle hypochlorite solution according to guidelines. 
	NOTE: When using RNAi to down regulate the gene of interest, synchronize the hermaphrodites as described in section 2, with the exception of the transfer of the mothers to RNAi plates seeded with the specific RNAi bacteria. When the knockdown with the RNAi is weak, feeding for multiple generations is recommended.</li>
</ol>
3. Performing the Oxidative Stress Resistance Assay
<ol>
	<li>Pipette 40 &#181;l of the 100 mM PQ solution (prepared fresh) to every well of a 96 well plate. Use at least 12 wells as replicates of every condition (treatment, mutant, etc.). Using a platinum wire worm pick, transfer 5-8 L4 larvae animals to every well indicating the start time and the end time.</li>
	<li>When starting another condition, note the start time and the end time. Place the plate at 20 &#176;C in the incubation time between transferring the worms and scoring dead animals an hour later from the start time.</li>
	<li>Using the dissecting microscope, count survival every hr. Gently shake the plate before starting to count. Indicate worms that do not move, even after spotting a high-intensity light, as dead animals. Also indicate the number of animals alive. 
	NOTE: Looking at worm shape, tails and head movement at high magnification, help determine dead worms from alive.</li>
	<li>Repeat this step every hour until most worms are dead.</li>
</ol>
4. Determination of Survival Percentage at Every Time Point
<ol>
	<li>Calculate the total number of animals per condition by adding the total number of animals transferred to every well. Ignore the animals that are damaged or killed upon transfer.</li>
	<li>For every time point, calculate the total number of dead animals per condition (sum of dead animals in the 12 wells). For every time point, calculate percentage of death (number of dead animals divided by total number of animals and multiplied by 100) and percentage of survival (100 minus percent death).</li>
	<li>Repeat this assay at least 3 independent times.</li>
</ol>

<ol>
	<li>Schieber, M., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+function+in+redox+signaling+and+oxidative+stress.">ROS function in redox signaling and oxidative stress.</a> Curr Biol. 24, R453-R462 (2014).</li><li>Alfadda, A. A., Sallam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+health+and+disease.">Reactive oxygen species in health and disease.</a> J Biomed Biotechnol. 2012, 936486 (2012).</li><li>Finkel, T., Holbrook, 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=Oxidants,+oxidative+stress+and+the+biology+of+ageing.">Oxidants, oxidative stress and the biology of ageing.</a> Nature. 408, 239-247 (2000).</li><li>Di Carlo, M., Giacomazza, D., Picone, P., Nuzzo, D., San Biagio, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+oxidative+stress+and+mitochondrial+dysfunction+the+key+players+in+the+neurodegenerative+diseases.">Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases.</a> Free Radic Res. 46, 1327-1338 (2012).</li><li>Gandhi, S., Abramov, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+oxidative+stress+in+neurodegeneration.">Mechanism of oxidative stress in neurodegeneration.</a> Oxid Med Cell Longev. 2012, 428010 (2012).</li><li>Trushina, E., McMurray, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+mitochondrial+dysfunction+in+neurodegenerative+diseases.">Oxidative stress and mitochondrial dysfunction in neurodegenerative diseases.</a> Neuroscience. 145, 1233-1248 (2007).</li><li>Touyz, R. M., Briones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+vascular+biology:+implications+in+human+hypertension.">Reactive oxygen species and vascular biology: implications in human hypertension.</a> Hypertens Res. 34, 5-14 (2011).</li><li>Gorrini, C., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+oxidative+stress+as+an+anticancer+strategy.">Modulation of oxidative stress as an anticancer strategy.</a> Nat Rev Drug Discov. 12, 931-947 (2013).</li><li>Sosa, 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=Oxidative+stress+and+cancer:+an+overview.">Oxidative stress and cancer: an overview.</a> Ageing research reviews. 12, 376-390 (2013).</li><li>Van Raamsdonk, J. M., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Oxygen+Species+and+Aging+in+Caenorhabditis+elegans:+Causal+or+Casual+Relationship.">Reactive Oxygen Species and Aging in Caenorhabditis elegans: Causal or Casual Relationship.</a> Antioxid Redox Signal. 13, 1911-1953 (2010).</li><li>Baumeister, R., Schaffitzel, E., Hertweck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+signaling+in+Caenorhabditis+elegans+controls+stress+response+and+longevity.">Endocrine signaling in Caenorhabditis elegans controls stress response and longevity.</a> J Endocrinol. 190, 191-202 (2006).</li><li>Markaki, M., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+in+Caenorhabditis+elegans.">Modeling human diseases in Caenorhabditis elegans.</a> Biotechnol J. 5, 1261-1276 (2010).</li><li>Rodriguez, M., Snoek, L. B., De Bono, M., Kammenga, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+under+stress:+C.+elegans+stress+response+and+its+relevance+to+complex+human+disease+and+aging.">Worms under stress: C. elegans stress response and its relevance to complex human disease and aging.</a> Trends Genet. 29, 367-374 (2013).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical approach series. , 282 (1999).</li><li>C. elegans Sequencing Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn+to+the+worm!.">Turn to the worm!.</a> Current opinion in genetics &amp; development. 7, 410-415 (1997).</li><li>Wheelan, S. J., Boguski, M. S., Duret, L., Makalowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+nematode+orthologs--lessons+from+the+analysis+of+1800+human+genes+and+the+proteome+of+Caenorhabditis+elegans.">Human and nematode orthologs--lessons from the analysis of 1800 human genes and the proteome of Caenorhabditis elegans.</a> Gene. 238, 163-170 (1999).</li><li>Culetto, E., Sattelle, D. 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+role+for+Caenorhabditis+elegans+in+understanding+the+function+and+interactions+of+human+disease+genes.">A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.</a> Hum Mol Genet. 9, 869-877 (2000).</li><li>Fire, A., 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=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+approaches+in+C.+elegans.">Functional genomic approaches in C. elegans.</a> Methods Mol Biol. 351, 127-138 (2006).</li><li>Poulin, G., Nandakumar, R., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screens+in+Caenorhabditis+elegans:+impact+on+cancer+research.">Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research.</a> Oncogene. 23, 8340-8345 (2004).</li><li>Moreno-Arriola, E., 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=Caenorhabditis+elegans:+A+Useful+Model+for+Studying+Metabolic+Disorders+in+Which+Oxidative+Stress+Is+a+Contributing+Factor.">Caenorhabditis elegans: A Useful Model for Studying Metabolic Disorders in Which Oxidative Stress Is a Contributing Factor.</a> Oxid Med Cell Longev. , 705253 (2014).</li><li>Possik, E., 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=Folliculin+regulates+ampk-dependent+autophagy+and+metabolic+stress+survival.">Folliculin regulates ampk-dependent autophagy and metabolic stress survival.</a> PLoS Genet. 10, e1004273 (2014).</li><li>Fukushima, T., Tanaka, K., Lim, H., Moriyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+cytotoxicity+of+paraquat.">Mechanism of cytotoxicity of paraquat.</a> Environ Health Prev Med. 7, 89-94 (2002).</li><li>Van Raamsdonk, J. M., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+is+dispensable+for+normal+animal+lifespan.">Superoxide dismutase is dispensable for normal animal lifespan.</a> Proc Natl Acad Sci U S A. 109, 5785-5790 (2012).</li><li>Schulz, T. J., 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=Glucose+restriction+extends+Caenorhabditis+elegans+life+span+by+inducing+mitochondrial+respiration+and+increasing+oxidative+stress.">Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress.</a> Cell Metab. 6, 280-293 (2007).</li><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, 71-94 (1974).</li><li>Timmons, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+methods+for+RNA+interference+in.">Delivery methods for RNA interference in.</a> C. elegans. Methods Mol Biol. 351, 119-125 (2006).</li><li>Wang, B. Y., 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=Caenorhabditis+elegans+Eyes+Absent+Ortholog+EYA-1+Is+Required+for+Stress+Resistance.">Caenorhabditis elegans Eyes Absent Ortholog EYA-1 Is Required for Stress Resistance.</a> Biochemistry (Mosc). 79, 653-662 (2014).</li><li>Paz-Gomez, D., Villanueva-Chimal, E., Navarro, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEAD+Box+RNA+helicase+VBH-1+is+a+new+player+in+the+stress+response+in+C.+elegans.">The DEAD Box RNA helicase VBH-1 is a new player in the stress response in C. elegans.</a> PLoS One. 9, 97924 (2014).</li><li>Ward, J. D., 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=Defects+in+the+C.+elegans+acyl-CoA+synthase,+acs-3,+and+nuclear+hormone+receptor,+nhr-25,+cause+sensitivity+to+distinct,+but+overlapping+stresses.">Defects in the C. elegans acyl-CoA synthase, acs-3, and nuclear hormone receptor, nhr-25, cause sensitivity to distinct, but overlapping stresses.</a> PLoS One. 9, 92552 (2014).</li><li>Staab, T. A., Evgrafov, O., Knowles, J. A., Sieburth, D. <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+synaptic+nlg-1/neuroligin+abundance+by+the+skn-1/Nrf+stress+response+pathway+protects+against+oxidative+stress.">Regulation of synaptic nlg-1/neuroligin abundance by the skn-1/Nrf stress response pathway protects against oxidative stress.</a> PLoS Genet. 10, e1004100 (2014).</li><li>Greer, E. 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=An+AMPK-FOXO+pathway+mediates+longevity+induced+by+a+novel+method+of+dietary+restriction+in.">An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in.</a> C. elegans. Curr Biol. 17, 1646-1656 (2007).</li><li>Allen, E., Walters, I. B., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brivanib,+a+dual+FGF/VEGF+inhibitor,+is+active+both+first+and+second+line+against+mouse+pancreatic+neuroendocrine+tumors+developing+adaptive/evasive+resistance+to+VEGF+inhibition.">Brivanib, a dual FGF/VEGF inhibitor, is active both first and second line against mouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance to VEGF inhibition.</a> Clin Cancer Res. 17, 5299-5310 (2011).</li><li>Apfeld, J., O'Connor, G., McDonagh, T., DiStefano, P. S., Curtis, 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+AMP-activated+protein+kinase+AAK-2+links+energy+levels+and+insulin-like+signals+to+lifespan+in+C.+elegans.">The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans.</a> Genes Dev. 18, 3004-3009 (2004).</li><li>Lee, H., 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=The+Caenorhabditis+elegans+AMP-activated+protein+kinase+AAK-2+is+phosphorylated+by+LKB1+and+is+required+for+resistance+to+oxidative+stress+and+for+normal+motility+and+foraging+behavior.">The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.</a> J Biol Chem. 283, 14988-14993 (2008).</li><li>Honda, Y., Honda, 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+daf-2+gene+network+for+longevity+regulates+oxidative+stress+resistance+and+Mn-superoxide+dismutase+gene+expression+in+Caenorhabditis+elegans.">The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.</a> FASEB J. 13, 1385-1393 (1999).</li><li>Restif, C., Metaxas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+swimming+motions+of+C.+elegans+worms+with+applications+in+aging+studies.">Tracking the swimming motions of C. elegans worms with applications in aging studies.</a> Med Image Comput Comput Assist Interv. 11, 35-42 (2008).</li><li>Buckingham, S. D., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+automated+measurement+of+nematode+swimming+(thrashing)+without+morphometry.">Fast, automated measurement of nematode swimming (thrashing) without morphometry.</a> BMC Neurosci. 10, 84 (2009).</li></ol>

The authors declare that they have no competing financial interests.

C. elegans is an attractive model organism to study genetically oxidative stress resistance in vivo since it can be easily cultured, and rapidly leads to a large number of genetically identical offspring. Multiple methods to measure oxidative stress resistance have been previously described and they are based on the supplementation of culture plates with various ROS sources such as PQ, rotenone, H2O2, and juglone25,26,29-32. Here we describe a protocol that measures oxidative stress resistance in liquid in 96 well plates using 100 mM PQ. A similar assay has been performed by Greer et al.33. This assay could be further adapted to study oxidative stress in liquid using other ROS sources. For instance, we have shown that loss of flcn-1 increases resistance to several H2O2 concentrations as well23.
This assay could be technically challenging for some strains that display motility defects or swimming-induced paralysis phenotypes such as strains carrying a mutation in the dopamine transporter dat-134. In this case, the inability to move is confusing since it does not necessarily mean decreased oxidative stress resistance but rather a motility defect. Furthermore, counting of dead animals should be carefully completed and researchers have to pay attention to C. elegans behavioral details such as tail movements, and head movements, or even slow body movements. If the PQ concentration is too high, and the death kinetics of the animals is very fast, one might consider decreasing the concentration of PQ in order to get slower death rates. Some animals are extremely sensitive to oxidative stress such as aak-2 mutant animals26,33,35,36 while others are highly resistant such as daf-2 mutant animals37. In this case, assaying survival with different concentrations of PQ should be considered.
For RNAi experiments, negative results could be due to an inefficiency of the RNAi treatment. In this case, measurement of mRNA levels is essential to determine whether the gene knockdown is successful.
This protocol could be further adapted for high throughput screening of oxidative stress resistance using a detector that tracks the swimming behavior of C. elegans in liquid38,39. This would potentially enable the monitoring of the endurance of C. elegans and the swimming behavior such as frequency of body bending under oxidative stress. Higher rates of body movement could indicate higher worm fitness under stress.
Pathways that lead to oxidative stress resistance in lower eukaryotes are highly conserved across evolution and are linked to oxidative stress-associated diseases and aging in humans. Mitohormesis and the balanced generation of ROS are essential to extend lifespan and improve health span. However, excessive ROS is highly damaging to cells, tissues/organs, and organisms. Finding pathways, that if altered, provide an optimal ROS balance is thus essential and the screens for drugs that modulate the resistance to oxidative stress could help the development of cures for multiple diseases with the common denominator: oxidative stress. Performing these screenings in C. elegans is an advantageous fast, inexpensive, and reliable method that has great potential and value for the understanding and treatment of human diseases linked to oxidative stress.

A reference was corrected in the publication of <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

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

In eukaryotes, oxidative phosphorylation taking place in the electron transport chain of the mitochondria is the main driver of energy production in the form of ATP. Reactive oxygen species (ROS) are a natural byproduct of this process. Despite their important role as signaling molecules, excessive ROS can lead to DNA damage, protein carbonylation, and lipid oxidation. An imbalance between ROS production and detoxification causes oxidative stress, which leads to energy depletion, cellular damage, and triggers cell death1,2. Oxidative stress contributes to aging and to the development of many life-threatening diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases3-9.
Cells have evolved enzymatic and non-enzymatic defense strategies to maintain proper ROS levels and to protect their constituents against oxidative damage1,2. Superoxide dismutase (SOD) enzymes act first to convert superoxide to H2O2, which is later converted to water by catalase or peroxidase enzymes. Non enzymatic defense strategies include mostly molecules that react faster with ROS as compared to cellular macromolecules, protecting essential cellular components. Despite the protective role of ROS detoxifying enzymes, some ROS molecules escape the antioxidant defense mechanisms and lead to oxidative damage. Detection, repair, and degradation of the damaged cellular components are essential defense strategies during oxidative stress1,2.
Signaling pathways involved in stress resistance and specifically oxidative stress are highly evolutionarily conserved10,11. Unlike cell culture experiments where organismal conditions are only partially reproduced, the study of oxidative stress in model organisms12,13 has great significance. C. elegans is a free-living nematode that can be easily and inexpensively cultured on a bacterial lawn on agar media. It is small in size (about 1 mm in length) and normally grows as a self-fertilizing hermaphrodite, which facilitates genetic manipulations. It has a rapid life cycle and a high reproductive capacity, producing about 300 offspring per generation, making it a powerful tool to perform large-scale genetic screens14. The C. elegans genome is fully sequenced and 40-50% of the genes are predicted to be homologues of human disease-associated genes15-18. The knockdown of genes of interest using RNAi is rapid and easy in C. elegans. Gene down regulation could be achieved by feeding animals the E. coli bacteria that harbor a plasmid expressing the double-stranded RNA that targets the mRNA of interest19. Therefore, determination of gene function using large scale RNAi screens has great impact on understanding human diseases including cancer 20,21.
Studies of oxidative stress resistance in C. elegans have led to the identification of conserved mechanisms of resistance to oxidative stress13,22. Some pathways identified are common pathways that modulate longevity and resistance to other stresses as well such as hypoxia, heat, and osmotic stress. These pathways include the insulin signaling, TOR signaling, and autophagy. Other key pathways involve detoxification of ROS such as superoxide dismutase enzymes and catalase enzymes, or in damage repair such as heat shock and chaperone proteins11,13,22.
This protocol describes how to determine the resistance to oxidative stress of C. elegans in liquid. We used flcn-1(ok975) and wild-type animals to demonstrate the protocol since we have previously shown an increased resistance to oxidative stress upon loss of flcn-1(ok975) in C. elegans23. We have also shown that this increased resistance depends on AMPK and autophagy, a signaling axis that improves cellular bioenergetics and promotes stress resistance 23. PQ is an oxidative stressor that interferes with the electron transport chain to produce reactive oxygen species24. The same assay could be adapted and other ROS sources or ROS generating compounds could be used such as H2O2 and rotenone. Similar assays have been developed on plates where low concentrations of PQ are used25,26. The advantage of this assay is that it is very fast, and the results could be obtained in one day. Additionally, the total volume of liquid used to perform the oxidative stress resistance assay in 96 well plates is low as compared to the volume used to prepare PQ-containing plates. Therefore, the amount of PQ used is in the liquid assay is low, which renders the assay inexpensive and limits the production of toxic wastes. However, limitations of this assay as compared to plate assays include the lack of food in the liquid assay and the lower concentration of oxygen in liquid as compared to air. These are important factors that in some cases, might influence the results. Therefore, confirming reproducibility using other methods of oxidative stress resistance is recommended to support results obtained in this assay.

<table><tbody><tr><td>Agar bacteriological grade</td><td>Multicell</td><td>800-010-LG</td><td></td></tr><tr><td>Bacteriological peptone</td><td>Oxoid</td><td>LP0037</td><td></td></tr><tr><td>Sodium chloride biotechnology grade</td><td>Bioshop</td><td>7647-14-5</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8503-25G</td><td></td></tr><tr><td>UltraPure tris hydrochloride</td><td>Invitrogen</td><td>15506-017</td><td></td></tr><tr><td>Tris aminomethane</td><td>Bio Basic Canada Inc</td><td>77-86-1</td><td></td></tr><tr><td>IPTG</td><td>Santa Cruz Biotechnology</td><td>sc-202185A</td><td></td></tr><tr><td>Ampicillin</td><td>Bioshop</td><td>69-52-3</td><td></td></tr><tr><td>Yeast extract</td><td>Bio Basic Inc.</td><td>8013-01-2</td><td></td></tr><tr><td>Methyl viologen dichloride hydrate</td><td>Aldrich chemistry</td><td>856177-1G</td><td></td></tr><tr><td>Petri dish 60 x15 mm</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Pipet 10 ml</td><td>Fisher</td><td>1367520</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>G-Biosciences</td><td>RC-084</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M-5921</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Bioshop</td><td>7558-79-4</td><td></td></tr><tr><td>Discovery v8 stereo zeiss microscope</td><td></td><td></td><td></td></tr><tr><td>96 well clear microtiter plate</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;flcn-1&lt;/em&gt; RNAi source</td><td>Ahringer Library</td><td></td><td></td></tr></tbody></table>

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.

Comparing wild-type C. elegans to flcn-1(ok975) mutant animals
Here we used 100 mM PQ to determine the resistance of wild-type C. elegans animals compared to flcn-1(ok975) which has been shown to resist oxidative stress, heat, and anoxia23. After 4 hr of treatment, 48.3% of wild-type survived as compared to 77.8% survival in flcn-1(ok975) animals. As expected, flcn-1(ok975) mutant animals were more resistant to 100 mM PQ compared to wild-type (Figure 2 and Table 1).
Comparing wild-type animals fed with control EV or flcn-1 RNAi bacteria
Similar to the result presented in the previous section, down regulation of flcn-1 using RNAi increased the resistance to 100 mM PQ. This result demonstrates that down regulation of gene function using RNAi mimics loss-of-function mutation (Figure 2 and Table 1).
<img alt="Figure 1" src="/files/ftp_upload/52746/52746fig1.jpg" /> 
Figure 1.&#160;Schematic figure of the method of oxidative stress resistance determination in 96 well plates in C. elegans.
Synchronized L4/young adult animals grown on MYOB plates and fed with E. coli OP50 bacteria are transferred to the wells of a 96 well microtiter plate containing 100 mM PQ and survival is measured hourly until a large number of worms are dead. In the case of RNAi knockdowns, the same procedure is followed except that the synchronized animals are grown on plates supplemented with IPTG, and are fed with the E. coli HT115 bacteria harboring the plasmid that will knockdown the target gene upon expression.
<img alt="Figure 2" src="/files/ftp_upload/52746/52746fig2.jpg" /> 
Figure 2.&#160;Loss of FLCN-1 increases resistance to oxidative stress in C. elegans. (A-B) Percent survival to 100 mM PQ of (A) w.t. and flcn-1(ok975) mutant animals and (B) wt animals treated with control or flcn-1RNAi.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td colspan="5">Percent survival to 100 mM PQ</td>
		</tr>
		<tr>
			<td>1 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>82.38 &#177; 1.31</td>
			<td align="right">0.0002</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>99.03 &#177; 1.67</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>91.70 &#177; 0.55</td>
			<td align="right">0.0462</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>95.93 &#177; 2.48</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>72.13 &#177; 7.13</td>
			<td align="right">0.005</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>96.33 &#177; 2.28</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>80.27 &#177; 5.34</td>
			<td align="right">0.0261</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>91.23 &#177; 1.42</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>55.37 &#177; 4.72</td>
			<td align="right">0.0004</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>87.00 &#177; 1.36</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>70.77 &#177; 3.50</td>
			<td align="right">0.0027</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>87.63 &#177; 2.67</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>48.30 &#177; 6.39</td>
			<td align="right">0.002</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>77.80 &#177; 3.12</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>63.77 &#177; 0.66</td>
			<td align="right">0.0122</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>80.57 &#177; 6.68</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>41.60 &#177; 8.33</td>
			<td align="right">0.0062</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>67.43 &#177; 1.42</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>60.53 &#177; 2.58</td>
			<td align="right">0.0313</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>71.53 &#177; 5.24</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6 hr</td>
			<td>Strain, RNAi</td>
			<td>Percent Survival (&#177;SD)</td>
			<td>P Value</td>
			<td>Number of experiments</td>
			<td>Total Number of worms</td>
		</tr>
		<tr>
			<td></td>
			<td>wt</td>
			<td>34.86 &#177; 5.88</td>
			<td align="right">0.0021</td>
			<td align="right">3</td>
			<td align="right">210</td>
		</tr>
		<tr>
			<td></td>
			<td>flcn-1(ok975)</td>
			<td>60.34 &#177; 2.05</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">224</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; control</td>
			<td>44.43 &#177; 3.93</td>
			<td align="right">0.0042</td>
			<td align="right">3</td>
			<td align="right">202</td>
		</tr>
		<tr>
			<td></td>
			<td>wt; flcn-1(RNAi)</td>
			<td>62.13 &#177; 3.44</td>
			<td></td>
			<td align="right">3</td>
			<td align="right">207</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Summary of results and statistical analysis of the oxidative stress resistance from Figure 2.

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

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.

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 ...

measuring-oxidative-stress-resistance-caenorhabditis-elegans-96-well

Nanyang Technological University

Research

JoVE Journal

Biology

15.4K Views.  McGill University. 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. 

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

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

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

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

Summary

Explore More Videos

Chapters in this video

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

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

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurements of Physiological Stress Responses in C. Elegans

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

C. elegans Survival Assay: A Short-Term Toxicity Test