We wish to thank S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, and H-C. Hsiao for helpful discussions. This work was funded by start-up funds from the TAMHSC Department of Molecular and Cellular Medicine. The compound scope and spinning disk were purchased with funds provided by the department and the TAMHSC Office of the Dean. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources. pRF4 (rol-6(su1006)) was a gift of A. Fire.

1. Preparation of DiI stain
<ol>
 <li>Prepare a stock solution of 20 mg/mL DiI (Biotium, Inc., Hayward, CA) in DMF. DiI is light sensitive, so protect DiI from light by wrapping in foil.</li>
 <li>Create a working dilution of DiI by adding 0.6 &mu;L DiI stock to 399.4 &mu;L M9 for each population. This should give a final working dilution of 30 &mu;g/mL DiI in M9. This can be scaled up for staining multiple populations simultaneously. Shield DiI from light by wrapping the tube(s) in foil.</li>
</ol>
2. Preparation of nematodes
<ol>
 <li>Use a 60 mm plate containing a population of uncontaminated nematodes. Wash animals from plate using a solution of 0.5% Triton X-100 in M9 buffer by gently swirling liquid in a circular motion across the surface of the plate to loosen all larval and adult animals. Transfer wash into a sterile 1.5 mL tube.</li>
 <li>Immediately spin down the animals at 2000 rpm for 30 sec. Remove and discard as much supernatant as possible without disturbing the mass of animals at the bottom of the tube.</li>
 <li>To reduce residual Triton X-100, rinse animals using M9 buffer, spin, and remove supernatant. Repeat this step once.</li>
 <li>Add 400 &mu;L of working DiI solution in M9 to the tube and vortex briefly to resuspend animals in the solution.</li>
 <li>Shake tube at 20&deg;C horizontally for 3 hours at 350 rpm in a light-protected environment. If desired, animals can be incubated up to 16 hours for staining.</li>
 <li>To reduce the amount of unbound dye, spin down the animals at 2000 rpm for 20 sec. Remove and discard as much supernatant as possible without disturbing the mass of animals.</li>
 <li>Resuspend animals in 400 &mu;L M9 buffer and pour liquid onto a bacteria-free portion of a NGM agar plate seeded with OP50 E. coli. Allow animals to recover at least 30 minutes. During the recovery time the animals should crawl away from the DiI staining liquid and onto the food. This step reduces background fluorescence from free DiI.</li>
</ol>


3. Mounting and observing specimens
<ol>
 <li>Melt 4% agar in water using an autoclave or a microwave.</li>
 <li>Create reusable spacers, which can be used to ensure uniform thickness of the agar pad, by layering two pieces of lab tape on a glass slide. Make two spacer slides total.</li>
 <li>Arrange a clean glass slide between two spacer slides. Pipette about 150 &mu;L (four drops) of molten 4% agar onto the center of the clean glass slide. Quickly cover the molten agar using an additional slide to form an agar pad. Carefully remove the cover slide, keeping the pad centered on the top of the mounting slide.</li>
 <li>Pipette about 5 &mu;L of nematode anesthetic (100 &mu;M - 1 mM levamisole, for example) onto the pad.</li>
 <li>Mount 8 - 12 animals in the anesthetic and cover with a microscope coverslip.</li>
 <li>Observe animals using a compound or confocal microscope fitted with at least a 40x objective and a DSRed/TRITC (or other compatible) filter. The fluorescence excitation maximum of DiI is 549 nm and its emission maximum is 565 nm for bound dye (Biotium, Inc., Hayward, CA).</li>
</ol>
4. Representative Results
DiI stains the cuticle of wild-type and mutant C. elegans. The cuticular surface contains annuli separated by circumferential furrows and, in some stages, longitudinal ridges called alae. Each developmental stage has cuticular structures with distinct compositions2. Ridges or furrows of both alae and annuli fluorescently stain, depending on surface composition, throughout larval and adult stages and remain visible up to a day after recovery using this method. Background fluorescent speckles are sometimes observed (Figures 2F, G), but not routinely (Figure 1, Figure 2A-E, H, I). All images were taken with spinning disk confocal or, when noted, widefield (wf) compound microscopy.
<img alt="Figure 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> 
Figure 1. DiI stains cuticle and environmentally exposed neurons. L2 stage animal. 630x magnification. Mosaic image parts were captured using iVision-Mac software (BioVision Technologies, Exton, PA). Images were joined using Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Scale bar = 10 &mu;m. DiI also fluorescently stains amphid and phasmid sensory neurons in the head and tail respectively (arrows mark some).
<img alt="Figure 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> 
Figure 2. DiI fluorescently stains C. elegans cuticle at all stages of post-embryonic development. 630x magnification. Scale bar = 10 &mu;m. Staining of wild-type animals in A) L1; B) L2; C) dauer; D) L3; E) L4; and F) adult stages. The alae and annular ridges are fluorescently stained in L1 and dauer animals (A, C). DiI stains annular ridges in L2 animals (B). Annular furrows stain in L3 and L4 animals (D, E). The furrows of the alae and annuli are stained in adult animals (F-H). Alae are composed of two, five, or three ridges (in L1, dauer, or adult animals, respectively) that run the length of the animal (arrows)16. Annuli create circumferential ridges around the animal (arrowheads, F and J). The cuticle of adult mutant animals display moderate cuticular organization defects (G-H). G) Ridges of alae are discontinuous (wf). H) Supernumerary alae ridges are fused and branched or bifurcated (wf). Collagen gene mutants exhibit alae and annular organization defects (I-J). I) In transgenic animals overexpressing pRF4 (rol-6(su1006)), ridges of alae lie at an angle to the length of the animal. J) Annuli in transgenic animals overexpressing pRF4 (rol-6(su1006)) display an irregular pattern. 
<img alt="Figure 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> 
Figure 3. External morphological structures are illuminated by DiI staining. 630x magnification. Scale bars = 10 &mu;m. DiI also highlights other exterior features, including A) adult hermaphrodite vulva, B) adult male tail rays and fan, and C) hermaphrodite tail spike (forked in this mutant background).
<img alt="Figure 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> 
Figure 4. Cuticle takes longer to stain with DiI than environmentally exposed neurons. 630x magnification. Scale bar = 10 &mu;m. After two hours of staining, amphid (not shown) and phasmid sensory neurons (arrows) are sufficiently stained. In contrast, the cuticle of younger animals is only partially stained in patches (arrowhead).
<table border="1">
 <tbody>
 <tr>
 <td>Wash</td>
 <td>Stain solution (+ DiI)</td>
 <td>Incubation time</td>
 <td>Cuticle stained</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>M9 + 0.5% Triton X-100</td>
 <td>2 hrs</td>
 <td>no</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>M9 + 0.5% Triton X-100</td>
 <td>3 hrs</td>
 <td>no</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>M9</td>
 <td>2 hrs</td>
 <td>partial</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>M9</td>
 <td>3 hrs</td>
 <td>yes</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>H20</td>
 <td>2 hrs</td>
 <td>partial</td>
 </tr>
 <tr>
 <td>M9 + 0.5% Triton X-100</td>
 <td>H20</td>
 <td>3 hrs</td>
 <td>yes</td>
 </tr>
 </tbody>
</table>
 
Table 1. Cuticular staining under different conditions. Various incubation solutions and times were tested to optimize cuticular staining in animals. H20, sterile distilled water. Partial staining indicates patchy staining of larval cuticle (Figure 4), though adult cuticle stains consistently.

<ol>
	<li>Cox, G. N., Kusch, M., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+of+Caenorhabditis+elegans:+its+isolation+and+partial+characterization.">Cuticle of Caenorhabditis elegans: its isolation and partial characterization.</a> The Journal of Cell Biology. 90, 7-17 (1981).</li><li>Cox, G. N., Staprans, S., Edgar, R. 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+cuticle+of+Caenorhabditis+elegans.+II.+Stage-specific+changes+in+ultrastructure+and+protein+composition+during+postembryonic+development.">The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development.</a> Dev. Biol. 86, 456-470 (1981).</li><li>Hall, D., Altun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans Atlas. , (2008).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> The C. elegans Research Community. , (2007).</li><li>Kramer, J. M., Johnson, J. J., Edgar, R. S., Basch, C., Roberts, 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+sqt-1+gene+of+C.+elegans+encodes+a+collagen+critical+for+organismal+morphogenesis.">The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis.</a> Cell. 55, 555-565 (1988).</li><li>Mende, N. v. o. n., Bird, D. M., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dpy-13:+a+nematode+collagen+gene+that+affects+body+shape.">dpy-13: a nematode collagen gene that affects body shape.</a> Cell. 55, 567-576 (1988).</li><li>Blaxter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+surface+proteins+of+wild+type+and+mutant+Caenorhabditis+elegans.">Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 268, 6600-6609 (1993).</li><li>Costa, M., Draper, B. W., Priess, 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=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+collagen+genes.+Expression+in+Caenorhabditis+elegans.">Cuticle collagen genes. Expression in Caenorhabditis elegans.</a> Trends Genet. 16, 21-27 (2000).</li><li>Kramer, J. M., French, R. P., Park, E. C., Johnson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+rol-6+gene,+which+interacts+with+the+sqt-1+collagen+gene+to+determine+organismal+morphology,+encodes+a+collagen.">The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.</a> Mol. Cell Biol. 10, 2081-2089 (1990).</li><li>Thein, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, 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=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</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></ol>

We have nothing to disclose.

The DiI staining method presented here allows for a relatively quick and convenient way to visualize the cuticle in C. elegans. By repurposing and optimizing a method commonly used to image environmentally exposed sensory neurons15,17, DiI can be used to fluorescently stain both alae and annular structures (Figures 1 and 2), as well as the vulva, male tail, and hermaphrodite tail spike (Figure 3). We have found that the incubation solution and time influence the ability of DiI to consistently stain th...

<table border="1">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Synonyms</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate</td>
			<td>Biotium, Inc.</td>
			<td>60010</td>
			<td>Stock dilution: 
			20 mg/mL in DMF 
			working dilution: 30 mg/mL</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Dimethylformamide</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>D4551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton 
			X-100</td>
			<td>Octylphenoxypolyethoxyethanol</td>
			<td>VWR International, LLC.</td>
			<td>EM-9410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M9</td>
			<td>22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1mM MgSO4</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NGM</td>
			<td>Nematode growth medium</td>
			<td>IPM Scientific, Inc.</td>
			<td>11006-501</td>
			<td>Can be prepared following NGM agar protocol18</td>
		</tr>
		<tr>
			<td>Agar-agar</td>
			<td>&nbsp;</td>
			<td>EMD Chemicals, Inc.</td>
			<td>1.01614</td>
			<td>4% in water</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Levamisole hydrochloride</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>31742</td>
			<td>100 &mu;M - 1 mM levamisole as required</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>&nbsp;</td>
			<td>VWR International, LLC.</td>
			<td>16005-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glasses</td>
			<td>&nbsp;</td>
			<td>VWR International, LLC.</td>
			<td>16004-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compound scope</td>
			<td>&nbsp;</td>
			<td>Carl Zeiss, Inc.</td>
			<td>A1m</td>
			<td>Use objectives to match the needs of the experiment</td>
		</tr>
		<tr>
			<td>TRITC or other compatible filter</td>
			<td>&nbsp;</td>
			<td>Chroma Technology Corp.</td>
			<td>49005</td>
			<td>ET - DSRed (TRITC/Cy3) sputtered filter set</td>
		</tr>
	</tbody>
</table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

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JoVE Journal

Biology

17.0K Views.  Texas A&M University System Health Science Center, College of Medicine. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Video: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

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 ...

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 ...

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

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

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

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

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...