Name: long-term culture and monitoring of isolated caenorhabditis elegans on solid media in multi-well devices
Uploaded: 2022-12-09T14:10:00
Description: Watch this Scientific Journal Video about Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices at JoVE.com

This work was supported by NIH R35GM133588 to G.L.S., a United States National Academy of Medicine Catalyst Award to G.L.S., the State of Arizona Technology and Research Initiative Fund administered by the Arizona Board of Regents, and the Ellison Medical Foundation.

1. Preparation of stock solutions and media
NOTE: Before beginning the preparation of the multi-well devices, prepare the following stock solutions and media.
<ol>
	<li>Stock solutions for nematode growth media (NGM) and low-melt NGM (lmNGM):
	<ol>
		<li>Prepare 1 M K2HPO4: Add 174.18 g of K2HPO4 to a 1 L bottle, and fill it up to 1 L with sterile deionized water. Autoclave (121 &#176;C, 15 psig) for 30 min, and store at room temperature (RT)...

<ol>
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(27), e1152 (2009).</li><li>Xian, B., 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=WormFarm:+A+quantitative+control+and+measurement+device+toward+automated+Caenorhabditis+elegans+aging+analysis.">WormFarm: A quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis.</a> Aging Cell. 12 (3), 398-409 (2013).</li><li>Rahman, 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=NemaLife+chip:+A+micropillar-based+microfluidic+culture+device+optimized+for+aging+studies+in+crawling+C.+elegans.">NemaLife chip: A micropillar-based microfluidic culture device optimized for aging studies in crawling C. elegans.</a> Scientific Reports. 10, 16190 (2020).</li><li>Chronis, N., Zimmer, M., Bargmann, C. 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P., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+swim+sessions+in+C.+elegans+induce+key+features+of+mammalian+exercise.">Single swim sessions in C. elegans induce key features of mammalian exercise.</a> BMC Biology. 15 (1), 30 (2017).</li><li>&#199;elen, &. #. 3. 0. 4. ;., Doh, J. H., Sabanayagam, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+liquid+cultivation+on+gene+expression+and+phenotype+of+C.+elegans.">Effects of liquid cultivation on gene expression and phenotype of C. elegans.</a> BMC Genomics. 19 (1), 562 (2018).</li><li>Pittman, W. 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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=Longitudinal+imaging+of+Caenorhabditis+elegans+in+a+microfabricated+device+reveals+variation+in+behavioral+decline+during+aging.">Longitudinal imaging of Caenorhabditis elegans in a microfabricated device reveals variation in behavioral decline during aging.</a> eLife. 6, 26652 (2017).</li><li>Jushaj, 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=Optimized+criteria+for+locomotion-based+healthspan+evaluation+in+C.+elegans+using+the+WorMotel+system.">Optimized criteria for locomotion-based healthspan evaluation in C. elegans using the WorMotel system.</a> PLoS One. 15 (3), 0229583 (2020).</li><li>Mittal, K. K., Mickey, M. R., Singal, D. P., Terasaki, P. 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Refinement of microdroplet lymphocyte cytotoxicity test.</a> Transplantation. 6 (8), 913-927 (1968).</li><li>Espejo, 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=Long-term+culture+of+individual+Caenorhabditis+elegans+on+solid+media+for+longitudinal+fluorescence+monitoring+and+aversive+interventions.">Long-term culture of individual Caenorhabditis elegans on solid media for longitudinal fluorescence monitoring and aversive interventions.</a> Journal of Visualized Experiments. , (2022).</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> Journal of Visualized Experiments. (64), e4019 (2012).</li><li>Freitas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worm+Paparazzi+-+A+high+throughput+lifespan+and+healthspan+analysis+platform+for+individual+Caenorhabditis+elegans.">Worm Paparazzi - A high throughput lifespan and healthspan analysis platform for individual Caenorhabditis elegans.</a> University of Arizona. , (2021).</li><li>Moore, B. T., Jordan, J. M., Baugh, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WormSizer:+High-throughput+analysis+of+nematode+size+and+shape.">WormSizer: High-throughput analysis of nematode size and shape.</a> PLoS One. 8 (2), e57142 (2013).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2013).</li><li>Roussel, N., Sprenger, J., Tappan, S. J., Glaser, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+tracking+and+quantification+of+C.+elegans+body+shape+and+locomotion+through+coiling,+entanglement,+and+omega+bends.">Robust tracking and quantification of C. elegans body shape and locomotion through coiling, entanglement, and omega bends.</a> Worm. 3 (4), 982437 (2014).</li><li>Grubbs, J. J., vander Linden, A. M., Raizen, D. 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The WorMotel system is a powerful tool for gathering individualized data for hundreds of isolated C. elegans over time. Following the earlier studies using multi-well devices for applications in developmental quiescence, locomotory behavior, and aging, the goal of this work was to optimize the preparation of multi-well devices for the long-term monitoring of activity, health, and lifespan in a higher-throughput manner. This work provides a detailed protocol for preparing multi-well devices that optimizes many of...

Caenorhabditis elegans are commonly used in aging studies because of their short generation time (approximately 3 days), short lifespan (approximately 3 weeks), ease of cultivation in the laboratory, high degree of evolutionary conservation of molecular processes and pathways with mammals, and wide availability of genetic manipulation techniques. In the context of aging studies, C. elegans allow for the rapid generation of longevity data and aged populations for the analysis of late-life phenotypes in live animals. The typical approach for conducting worm aging studies involves manually measuring the lifespan of a population of worms maintained in groups of 20 to 70 animals on solid agar nematode growth media (NGM) in 6 cm Petri plates1. Using age-synchronized populations allows the measurement of lifespan or cross-sectional phenotypes in individual animals across the population, but this method precludes monitoring the characteristics of individual animals over time. This approach is also labor-intensive, thus restricting the size of the population that can be tested.
There are a limited number of culture methods that allow for the longitudinal monitoring of individual C. elegans throughout their lifespan, and each has a distinct set of advantages and disadvantages. Microfluidics devices, including WormFarm2, NemaLife3, and the &#34;behavior&#34; chip4, among others5,6,7, allow the monitoring of individual animals over time. Culturing worms in liquid culture using multi-well plates similarly allows the monitoring of either individual animals or small populations of C. elegans over time8,9. The liquid environment represents a distinct environmental context from the common culture environment on solid media in Petri plates, which can alter aspects of animal physiology that are relevant to aging, including fat content and the expression of stress-response genes10,11. The ability to directly compare these studies to the majority of data collected on aging C. elegans is limited by differences in potentially important environmental variables. The Worm Corral12 is one approach developed to house individual animals in an environment that more closely replicates typical solid media culture. The Worm Corral contains a sealed chamber for each animal on a microscope slide using hydrogel, allowing the longitudinal monitoring of isolated animals. This method uses standard brightfield imaging to record morphological data, such as body size and activity. However, animals are placed in the hydrogel environment as embryos, where they remain undisturbed throughout their lifespan. This requires the use of conditionally sterile mutant or transgenic genetic backgrounds, which limits both the capacity for genetic screening, as each novel mutation or transgene needs to be crossed into a background with conditional sterility, and the capacity for drug screening, as treatments can only be applied once to the animals as embryos.
An alternative method developed by the Fang-Yen lab allows the cultivation of worms on solid media in individual wells of a microfabricated polydimethylsiloxane (PDMS) device called a WorMotel13,14. Each device is placed into a single-well tray (i.e., with the same dimensions as a 96-well plate) and has 240 wells separated by a moat filled with an aversive solution to prevent the worms from traveling between wells. Each well can house a single worm for the duration of its lifespan. The device is surrounded by water-absorbing polyacrylamide gel pellets (referred to as &#34;water crystals&#34;), and the tray is sealed with Parafilm laboratory film to maintain the humidity and minimize the desiccation of the media. This system allows healthspan and lifespan data to be gathered for individual animals, while the use of solid media better recapitulates the environment experienced by animals in the vast majority of published C. elegans lifespan studies, thus allowing more direct comparisons. Recently, a similar technique has been developed using polystyrene microtrays that were originally used for microcytotoxicity assays15 in place of the PDMS device16. The microtray method allows for the collection of individualized data for worms cultured on solid media and has improved capacity for containing worms under conditions that would typically cause fleeing (e.g., stressors or dietary restriction), with the trade-off being that each microtray can only contain 96 animals16, whereas the multi-well device utilized here can contain up to 240 animals.
Presented here is a detailed protocol for preparing multi-well devices that is optimized for plate-to-plate consistency and the preparation of multiple devices in parallel. This protocol was adapted from the original protocol from the Fang-Yen laboratory13. Specifically, there are descriptions for techniques to minimize contamination, optimize the consistent drying of both the solid media and the bacterial food source, and deliver RNAi and drugs. This system can be used to track individual healthspan, lifespan, and other phenotypes, such as body size and shape. These multi-well devices are compatible with existing high-throughput systems to measure lifespan, which can remove much of the manual labor involved in traditional lifespan experiments and provide the opportunity for automated, direct longevity measurement and health tracking in individual C. elegans at scale.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.5 lb weight</td>
			<td>CAP Barbell</td>
			<td>RP-002.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic sheets (6 in x 4 in x 3/8 in)</td>
			<td>Falken Design</td>
			<td>ACRYLIC-CL-3-8/1224</td>
			<td>Large sheet cut to smaller sizes&#160;</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9518</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable squeeze bottle</td>
			<td>Nalgene</td>
			<td>2405-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD Difco</td>
			<td>214030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>Thermo Scientific</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Basin, 25 mL</td>
			<td>VWR</td>
			<td>89094-664</td>
			<td>Disposable pipette basin&#160;</td>
		</tr>
		<tr>
			<td>Cabinet style vacuum desiccator&#160;</td>
			<td>SP Bel-Art</td>
			<td>F42400-4001</td>
			<td>Do not need to use dessicant, only using as a vacuum chamber.&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros Organics</td>
			<td>349615000</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>N2</td>
			<td>Wildtype strain</td>
		</tr>
		<tr>
			<td>Carbenicillin&#160;</td>
			<td>GoldBio</td>
			<td>C-103-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>360902</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>ICN Biomedicals Inc</td>
			<td>101380</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed oxygen tank</td>
			<td>Airgas</td>
			<td>UN1072</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Fisher Chemical</td>
			<td>C493-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bead bath incubator</td>
			<td>Fisher Scientific</td>
			<td>11-718-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50&#160;</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>OP50</td>
			<td>Standard labratory food for C. elegans</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore</td>
			<td>ex0276-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Floxuridine</td>
			<td>Research Products International</td>
			<td>F10705-1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Techne</td>
			<td>731-0177</td>
			<td>Used to cure PDMS mixture, any similar oven will suffice</td>
		</tr>
		<tr>
			<td>Incubators</td>
			<td>Shel Lab</td>
			<td>2020</td>
			<td>20 &#176;C incubator for maintaining worm strains and 37 &#176;C incubator to grow bacteria&#160;</td>
		</tr>
		<tr>
			<td>Isopropyl &#223;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>GoldBio</td>
			<td>I2481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher Chemical</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Chemical</td>
			<td>P286-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>KimTech</td>
			<td>34155</td>
			<td>Task wipes</td>
		</tr>
		<tr>
			<td>LB Broth, Lennox</td>
			<td>BD Difco</td>
			<td>240230</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melt agarose</td>
			<td>Research Products International</td>
			<td>A20070-250.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Chemical</td>
			<td>M-8900</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave&#160;</td>
			<td>Sharp</td>
			<td>R-530DK</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel repeat pipette, 20&#8211;200 &#181;L LTS EDP3</td>
			<td>Rainin</td>
			<td>17013800</td>
			<td>The exact model used is no longer sold, a similar model&#39;s catalog number has been provided</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc OmniTray</td>
			<td>Thermo Scientific</td>
			<td>264728</td>
			<td>Clear polystyrene trays</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Fisher Scientific</td>
			<td>13-374-10</td>
			<td>Double-wide (4 in)</td>
		</tr>
		<tr>
			<td>Petri plate, 100 mM&#160;</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate, 60 mM&#160;</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Plasma Etch, Inc.</td>
			<td>PE-50</td>
			<td></td>
		</tr>
		<tr>
			<td>PLATINUM vacuum pump</td>
			<td>JB Industries</td>
			<td>DV-142N&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet 3D printer</td>
			<td>Stratasys&#160;</td>
			<td>Objet500 Connex3</td>
			<td>PolyJet 3D printing services provided by ProtoCAM (Matrial: Vero Rigid; Finish: Matte; Color: Gloss; Resolution: X-axis: 600 dpi, Y-axis: 600 dpi, Z-axis: 1600 dpi)</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Lab-Line</td>
			<td>3526CC</td>
			<td></td>
		</tr>
		<tr>
			<td>smartSpatula</td>
			<td>LevGo, Inc.</td>
			<td>17211</td>
			<td>Disposable spatula</td>
		</tr>
		<tr>
			<td>Superabsorbent polymer (AgSAP Type S)</td>
			<td>M2 Polymer Technologies</td>
			<td>Type S</td>
			<td>Referred to in main text as &#34;water crystals&#34;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer base</td>
			<td>The Dow Chemical Company</td>
			<td>2065622</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer curing agent</td>
			<td>The Dow Chemical Company</td>
			<td>2085925</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.22 &#181;m)</td>
			<td>Nest Scientific USA Inc.&#160;</td>
			<td>380111</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL&#160;</td>
			<td>Fisher Scientific</td>
			<td>14955453</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Thermo Scientific</td>
			<td>J20605-AP</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Vacuum pump oil</td>
			<td>VWR</td>
			<td>54996-082</td>
			<td></td>
		</tr>
		<tr>
			<td>VeroBlackPlus</td>
			<td>Stratasys&#160;</td>
			<td>RGD875</td>
			<td>Rigid 3D printing filament</td>
		</tr>
		<tr>
			<td>Weigh boat</td>
			<td>Thermo Scientific</td>
			<td>WB30304</td>
			<td>Large enough for PDMS mixture volume</td>
		</tr>
	</tbody>
</table>

long-term culture and monitoring of isolated caenorhabditis elegans on solid media in multi-well devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

The WorMotel culture system can be used to gather a variety of data, including regarding lifespan, healthspan, and activity. Published studies have utilized multi-well devices to study lifespan and healthspan13,14, quiescence and sleep22,23,24, and behavior25. Lifespan can be scored manually or through a collection of images and downstream imaging...

Watch this Scientific Journal Video about Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices at JoVE.com

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

When we're studying aging and sea elegans we're typically following a population of animals over time. This gives us a glimpse of what that process you're studying is doing in the population at each time point but it doesn't tell you how the process is changing in individual animals over time. These single animal culture environments allow us to follow dynamic changes in molecular processes and physiological processes in individual animals over time for hundreds of animals in parallel.We find that there are two major things that can really impact the quality of these multi-well culture systems. One is either over or under drying the wells and the second is contamination. And we try to note throughout the protocol specific steps where you can try to mitigate these problems.Demonstrating the protocol today will be Emily Gardea. She's a technician in my lab and has done a lot of work to optimize this protocol. After preparing all the reagents, mix 60 grams of PDMS base and six grams of curing agent per mold in a large disposable way boat using a disposable spatula.Place the PDMS mixture in a vacuum chamber for 30 minutes at minus 0.08 Mega Pascal to remove bubbles. Pour and fill the PDMS mixture into each 3D printed mold. Put the filled mold and any extra PDMS mixture in the vacuum chamber for 25 minutes at minus 0.08 Mega Pascal.Remove the filled mold from the vacuum chamber and pop any remaining bubbles with a disposable spatula. Starting with one edge, slowly lower a flat acrylic sheet on top of the PDMS filled mold. If bubble formation occurs proceed as described in the manuscript.Place a 2.5 pound weight on the acrylic and place the weighted molds in the oven. Let them cure at 55 degrees Celsius overnight. At the end of the incubation, remove the weights and the molds from the oven.Next, work a razor blade between the acrylic and the cured PDMS To break the seal Using a razor or a metal spatula, carefully loosen the sides of the cured PDMS and remove them from the mold. Wrap the PDMS device in aluminum foil. Seal it with autoclave tape and sterilize by autoclaving on a dry cycle for at least 15 minutes at 121 degrees Celsius and 15 pounds per square inch gauge pressure.To prepare the multi-well device, preheat a dry bead bath incubator to 90 degrees Celsius. Unwrap an autoclaved multi-well device and cut a small notch in the top right corner of the device. Place up to three unwrapped devices into the plasma cleaner with the wells facing up and run the plasma cleaner.Sterilize one single well polystyrene tray per device by spraying the inside of the tray with 70%ethanol and wiping it dry with a task wipe. Remove each device from the plasma cleaner with a gloved hand and place it in a cleaned tray. Then place a 25 milliliter disposable pipette basin in the bead bath incubator preheated at 90 degrees Celsius.Take one tube of solidified LMNGM pre-mixture per device to be filled and place it in a 200 milliliter glass beaker. Remove the cap and Parafilm and microwave until the media melts sufficiently to pour. Pour the molten LMNGM pre-mixture into another sterile 200 milliliter beaker.Multiple test tubes can be combined in this beaker if more than one device is filled at a time. Microwave the LMNGM pre-mixture for an additional 20 seconds. If the pre-mixture begins to boil over stop the microwave and allow it to settle.Remove the molten LMNGM pre-mixture from the microwave and cool it to 60 degrees Celsius. Add the remaining LMNGM ingredients to the pre-mix mixture and pour the molten LMNGM into the 25 milliliter basin in the bead bath. Fill the device wells using a 200 microliter multi-channel repeater pipette.Set the repeater to dispense aliquots of 14 microliters and mount five pipette tips. Load the tips with molten LMNGM and dispense the first 14 microliters back into the LMNGM containing basin. Moving quickly but carefully, dispense 14 microliters into the inner wells of the device and dispense the remaining LMNGM back into the basin as the final aliquot is typically less than 14 microliters.Next, set the repeater to dispense aliquots of 15 microliters and fill the outermost ring of wells. Using a 200 microliter pipette, dispense 200 microliters of copper sulfate solution into the device moat in each corner. Avoid overfilling the moat and ensure that the copper sulfate does not touch the top surface of the wells.If the copper sulfate does not flow easily through the entire moat, use a short platinum wire pick to help break the tension and drag the copper sulfate through the moat. After the copper sulfate has flowed through the entire moat, remove as much copper sulfate as possible using a 200 microliter pipette or aspiration with a vacuum. Using a squeeze bottle, add the water crystals in the spaces between the device and the tray walls.Close the tray lid and wrap all four sides with a piece of Parafilm. Add two additional Parafilm pieces to seal the plate completely. Using a repeater pipette, spot each well with five microliters of concentrated bacteria.Avoid direct contact with the LMNGM surface. Dry the devices using a custom-built drying box which can be constructed at minimal cost using computer case fans and HEPA filters. Under a dissection scope, inspect wells to ensure all wells have dried successfully.Using a platinum pick, transfer the animals from the prepared worm plates and add one worm per well. Add a drop of prepared detergent solution to the inside of the tray lid and rub with a task wipe until the detergent solution has dried. Wrap the tray with three pieces of Parafilm as described in the manuscript.The lifespan extension from daf-2(RNAi)is blocked by the daf-16 null mutation. The health span also decreased in the presence of daf-2(RNAi)The three-day rolling mean of activity across the lifespan is reduced by daf-16 mutant and daf-2(RNAi)The variation in lifespan and health span across individual animals compared in either absolute terms or as a fraction of the total lifespan is shown. The cumulative activity across the lifespan for individual animals correlates better with lifespan than the activity for individual animals on any specific day across the lifespan.It's important to fill the wells and to add the worms as quickly as possible. Otherwise the wells can dry out and the worms won't survive. This system is compatible with automated imaging which can be used to gather high throughput data such as lifespan, activity and body size or shape.

long-term-culture-monitoring-isolated-caenorhabditis-elegans-on-solid

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...