D.S.A acknowledges funding from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq/Brazil), grant number #301808/2018-0, #313117/2019-5, Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Rio Grande do Sul (FAPERGS/Brazil), grant number, 21/2551-0001963-8, Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES, Finance Code 001 for N.S.J and A.C.S)

NOTE: Escherichia coli OP50 (E. coli OP50) bacteria and Bristol N2 wild type strains can be obtained from the CGC, University of Minnesota, USA or from donation from a C. elegans laboratory. For researchers&#39; safety, it is imperative to use Personal Protective Equipment. Although the concentrations of reagents like hypochlorite and sodium hydroxide are low, it is essential to wear the recommended PPE, as highlighted in the manuscript, to minimize any potential risks associated with these chemicals.
1. Glycogen content
<ol>
	<li>Preparation of test plates
	<ol>
		<li>Prepare six, 60 mm x 15 mm NGM agar plates20&#160;(10 mL of nematode growth media agar, NGM, as described in Supplementary Table 1) per worm strain and allow them to dry for 1 day at room temperature. Keep the plates closed to prevent contamination.</li>
		<li>Add 200 &#181;L of E. coli OP50 liquid culture (with an optical density = 0.600, approximately at a wavelength of 600 nm) per plate to inoculate a total of four, 60 mm x 15 mm NGM plates per strain in the flow hood. Allow the closed plates to incubate at 37 &#176;C for 1 day before use. 
		NOTE: Prepare six plates (step 1.1) but inoculate only 4 with E. coli OP50 bacteria. Reserve the remaining two plates at 4 &#176;C for later use when placing the worms under starvation conditions. This analysis may be performed in duplicate.</li>
		<li>On the test day, add 200 &#181;L of 0.025 M D-Glucose to two (6 mm x 15 mm) NGM agar plates per strain that were previously seeded with E. coli OP50 bacteria. Allow the plates to dry at room temperature in the flow hood. 
		NOTE: The NGM agar plates seeded with E. coli OP50 bacteria can also be dried near to alcohol lamps as an alternative to using a flow hood.</li>
	</ol>
	</li>
	<li>Synchronization
	<ol>
		<li>Approximately 3 days before the synchronization, transfer 3-4 chunks (3 cm x 3cm) from a maintenance NGM agar with C. elegans Bristol N2 (wild type) at different stages (around 500 worms per chunk) to a new plate (150 mm x 90 mm) that has been previously seeded with E. coli OP50 bacteria.</li>
		<li>Place the new plate with worms in a controlled environment at 20 &#176;C and maintain humidity &#62;95% for 3 days (72 h allow most of the worms to reach the gravid adult stage). 
		NOTE: The growth temperature may need to be adjusted depending on the C. elegans strain(s) being used.</li>
		<li>Using a Pasteur pipette, collect the worms from the prepared plate with distilled H2O and transfer them to a 50 mL centrifuge tube.</li>
		<li>Wait for the sedimentation of the worms by gravity (approximately 15 min) and then remove the supernatant. Repeat this process 3x to eliminate the bacteria. After the last wash, reduce the volume to 5 mL.</li>
		<li>Add 10 mL of bleaching solution (Supplementary Table 1)&#160;and vigorously shake using the hands for approximately 6 min.</li>
		<li>Immediately after the shaking is complete, fill the tubes to 50 mL capacity with M9 buffer.</li>
		<li>Centrifuge at 1400 x g for 3 min, remove the supernatant up to 5 mL and then add 45 mL of M9 buffer again. Repeat this process 4x.</li>
		<li>After the last wash, reduce the volume to 15 mL and maintain it at a controlled temperature (20 &#176;C) and humidity (&#62;95%) for approximately 14 h. 
		NOTE: The synchronization process can be replaced by obtaining eggs through oviposition: Transfer approximately 20 adult pregnant worms to an NGM plate (6 mm x 15 mm) previously seeded with 200 &#181;L of E. coli OP50. Allow the worms to lay eggs for 1 day. After that, remove the pregnant worms and wait for the eggs to hatch.</li>
	</ol>
	</li>
	<li>Preparation of worms
	<ol>
		<li>Using an automatic pipette add 10 &#181;L of the synchronized worms (step 1.2.8) to a microscope slide and count the number of worms. Calculate how much volume must be pipetted to obtain 500-1000 worms/&#181;L.</li>
		<li>Transfer 500-1000 L1 synchronized worms (step 1.2.8) to NGM agar plates (150 mm x 90 mm, Supplementary Table 1) seeded with E. coli OP50&#160;bacteria as a food source, until they reach the larvae stage 4 (L4) at 20 &#176;C. 
		NOTE: Worms can be transferred using an automatic pipette. If an automatic pipette is not available, use a glass Pasteur pipette, and volumes can be controlled using microtube labels or glass graduated pipettes.</li>
		<li>After 48 h of synchronization20: collect the L4 larvae from the plates with M9 buffer (Supplementary Table 1), with the aid of a plastic Pasteur pipette to a clean conical tube 50 mL and wash them 3x with fresh M9 buffer or repeat the washes until all remaining E. coli OP50 bacteria are completely removed (at room temperature). 
		NOTE: The washing step is important because some worms will experience starvation during the procedure.</li>
	</ol>
	</li>
	<li>Running the assay
	<ol>
		<li>Transfer 500-1000 L4 worms (step 1.3.1) from each of the new NGM agar plates (60mm x 15mm) previously prepared (steps 1.1.2 and 1.1.3). Keep them at 20 &#176;C for 48 h until the test day. This schedule will result in three experimental groups as following, in duplicate (Figure 1): 
		A- (starvation): worms will grow on regular NGM agar plates without E. coli OP50 bacteria; 
		B- (regular): worms will grow on regular NGM agar plates seeded with E. coli OP50 bacteria; 
		C- (high glucose): worms will grow on NGM agar plates seeded with E. coli OP50 bacteria and containing 0.025 M D-glucose.</li>
		<li>On the test day (after 48 h): collect the worms and give them three brief washes in the M9 buffer (steps 1.2.2 and 1.2.3) 
		NOTE: Here, prepare NGM agar plates with the addition of D-glucose (step 1.3) and set them aside until it is time to add the worms.
		<ol>
			<li>Iodine staining: prepare a 5% (v/v) Lugol&#39;s Iodine solution (iodine/potassium iodide solution) using fresh M9 buffer. 
			NOTE: Lugol&#39;s Iodine solution can be obtained from local reagent suppliers or pharmacies, therefore the initial concentration may vary. If necessary, calculate to obtain a 5% (v/v) solution.</li>
		</ol>
		</li>
		<li>Transfer approximately 100 &#181;L of washed worms (step 4.2) from each group to the 1.5 mL microtube containing diluted Lugol solution (5% v/v). Using an automatic pipette in a 1:5 proportion transfer 100 &#181;L of worms to 400 &#181;L of Lugol solution, followed by gentle agitation in a mixer for 5 min. 
		NOTE: If a mixer is unavailable, it is possible to shake the microtubes carefully by hands.</li>
		<li>Right after these 5 min, centrifuge the 1.5 mL microtube (worms + Lugol solution) at 1400 x g for 3 min. 
		NOTE: If this assay is performed on worms from the L4 stage or beyond, it is possible to achieve gravity sedimentation instead of using a centrifuge. Leave the microtubes open in racks on the benchtop for 10 min until all the worms have settled at the bottom of the microtube.</li>
		<li>Remove the supernatant and wash the worms with 1.0 mL of fresh M9 buffer. Repeat the wash until all remaining iodine is removed from the solution (minimum 3x).</li>
		<li>After the last washing step, remove the supernatant except for a residual of approximately 100 &#181;L. Use this to gently resuspend the worms and for microscopic analysis.</li>
		<li>Transfer approximately 50 &#181;L of the resuspended worm solution to microscopy slides and cover them with cover slips. Observe the bright field images using a stereomicroscope (at 1.5x). 
		NOTE: If a stereomicroscope is unavailable, a regular microscope can be used, and images can be captured with a cell phone camera (at 3.4x) using an adapter. To prevent errors from light/brightness/exposure, it is crucial that all cell phone camera settings remain consistent for all pictures.</li>
	</ol>
	</li>
	<li>Data inspection
	<ol>
		<li>Quantitative data: Calculate the glycogen content based on the iodine staining of worms.
		<ol>
			<li>Save images from stereomicroscope containing a minimum of 10 worms per group as a .jpeg file for further processing. Download the free ImageJ software to do this. Details are provided in Supplementary File 1, Supplementary Figure 1, Supplementary Figure 2.</li>
			<li>Open the .jpeg image using ImageJ software (which can be downloaded for free at https://imagej.nih.gov/ij/download.html). Click on&#160;&#8203;Segmented line and outline the worm (one worm at a time). Click on Analyze, and then select Measure to obtain the stain quantification. The data will be displayed as the Mean in the table. Mean indicates the data calculated from staining density per worm area.</li>
		</ol>
		</li>
		<li>Qualitative data: Obtain images from the stereomicroscope of each group and visually compare the staining of the worms. Create a scoring system, if needed, to analyze the staining: 0 colorless; 1 lightly stained; 2 stained and 3 heavily stained. Using the stereomicroscope, manually count approximately 10 worms per group (minimum) with a hand counter.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66084/66084fig01.jpg" /> 
Figure 1: Overall glycogen content assay schematic in C. elegans. A schematic of the experimental performed here to carry out the glycogen content assay. <a href="https://www.jove.com/files/ftp_upload/66084/66084fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Evaluation of intestinal permeability
<ol>
	<li>Preparation of worms
	<ol>
		<li>Approximately 14 h after synchronization (step 1.2.8), transfer 500-1000 worms (step 1.3.1 and NOTE) at the first larval stage (L1) to each new NGM agar plate (60 mm x 15 mm) previously prepared and keep them at 20 &#176;C until the day of the test. This schedule will lead to two experimental groups as follows (Figure 2): 
		A- (young): worms will grow on NGM agar plates seeded with the bacteria E. coli OP50 until larval stage 4 (L4); 
		B- (old): the worms will grow on NGM agar plates seeded with the bacteria E. coli OP50 until the 7th day of adulthood. 
		NOTE: The worms maintained until the 7th day of adulthood must be washed with M9 buffer every day and transferred to new NGM agar plates (60 mm x 15mm) with 200 &#181;L of E. coli OP50 bacteria previously seeded for food replacement and removal of the progeny. Younger worms float during decantation and can be separated by removing supernatant. The proportion of smurf worms increases with age. However, the evaluation of intestinal permeability can be performed at any stage and age, depending on the purpose of the study.</li>
	</ol>
	</li>
	<li>Running the assay
	<ol>
		<li>On the test day, collect L4 worms (2 days after step 2.1.1) and adulthood worms on 7th day (now in their 9th day) with M9 buffer (Supplementary Table 1) and transfer to labeled 1.5 mL microtubes.</li>
		<li>Centrifuge at 1400 x g for 3 min, remove the supernatant, add 1.0 mL of M9, and mix gently. Repeat this process 3x to remove the bacteria. After the last wash, reduce the volume to 500 &#181;L.</li>
		<li>Using an automatic pipette, add 10 &#181;L of the worm sediment to a microscope slide and count the number of worms. Calculate the volume to be pipetted to obtain 100 worms/&#181;L.</li>
		<li>Smurf staining: prepare a 25% Erioglaucine disodium salt (Supplementary Table 1) solution using distilled water. If necessary, use the vortex for better solubilization.</li>
		<li>In new previously identified microtubes, using an automatic pipette, add the volume containing 100 worms, 100 &#181;L of 25% Erioglaucine disodium salt solution, 200 &#181;L of E. coli OP50 and complete with M9 buffer for a final volume of 500 &#181;L. 
		NOTE: The volumes of Erioglaucine disodium, E. coli and number of worms are fixed. The volume of the M9 buffer is variable and can be used to bring the final volume to 500 &#181;L.</li>
		<li>Incubate for 3 h with agitation in a mixer, protected from light, at room temperature. 
		NOTE: If a mixer is unavailable, shake the microtubes carefully with your hands every 15 min.</li>
		<li>After 3 h, centrifuge the 1.5 mL microtube at 1400 x g for 3 min. Remove 1.0 mL of supernatant and wash the worms with 1.0 mL of M9 buffer.</li>
		<li>Repeat washes until the remaining Erioglaucine disodium salt solution is removed from the solution. After the last washing step, remove the supernatant except for a residual of approximately 250 &#181;L. Use this to carefully resuspend worms and process them for microscopic analysis.</li>
		<li>Transfer approximately 50 worms of resuspended worm solution to microscopy slides and cover them with cover slips. Incubate the microscopy slides in the refrigerator at -20 &#176;C for 10 min to paralyze the worms. 
		NOTE: Another alternative to paralyze the worms is to add 10 &#181;L of Levamisole hydrochloride solution (10 mM ; Supplementary Table 1) and cover them with cover slips.</li>
		<li>Observe and count the total number of worms and the totally dyed worms using the bright field in a stereomicroscope (at 1.5x). Images can be obtained from a cell phone camera (at 3.4x) coupled with an adapter. To prevent errors from light/brightness/exposure, it is crucial that all cell phone camera settings remain consistent for all pictures.</li>
	</ol>
	</li>
	<li>Data inspection
	<ol>
		<li>Qualitative data: Obtain images from stereomicroscope of each group (L4 and 7th day of adulthood) and visually compare the staining of the worms.</li>
		<li>Count the total number of worms and the number of smurf worms (blue worms) using a stereomicroscope.</li>
		<li>Express results as the percentage of smurf worms by comparing the young worms (L4, as a control group) and old worms (7th day of adulthood): 
		A x X = 100 (%) x B 
		X = 100 x B / A 
		Where, A = total number of worms 
		B= number of smurf worms</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66084/66084fig02.jpg" /> 
Figure 2: Overall intestinal permeability assay schematic in C. elegans. (A) C. elegans preparation. (B) Staining with Erioglaucine disodium salt. <a href="https://www.jove.com/files/ftp_upload/66084/66084fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Evaluating Metabolism and Aging Pattern in Caenorhabditis elegans

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The authors have nothing to disclose.

In summary, this protocol provides a qualitative evaluation of glycogen content in individual C. elegans worms using Lugol staining: a straightforward, robust, and swift assay. Lugol staining is a label-free and non-invasive approach that facilitates the acquisition of molecular data at subcellular resolutions, allowing for the monitoring of glycogen content fluctuations within single worms10. Furthermore, the assay offers the advantage of cost-effectiveness due to its minimal equipment r...

Biological sciences teaching in high school is a continuous challenge. Notably, the access and use of technology have brought important advances in the teaching-learning process, however, tools such as artificial intelligence chatbots make rationalizing and seeking evidence more difficult due to easy (and sometimes incorrect) responses obtained1. Because of that, the use of a scientific method with practical experimentation in an inquiry-based approach in the classroom is an important strategy to develop or stimulate critical thinking, creativity, and technical skills in the students2.
In this context, the free living nematode Caenorhabditis elegans has been successfully used in experimentation for teaching purposes3 because of its particular advantages: It is not a parasite and the Escherichia coli used for feeding is biosafety level-1, therefore reducing near to zero the biological hazard; it has an elegant and quantifiable locomotion movement, which is interesting for students to observe; and it is transparent, which allows organ observation, but also staining with pigments that can indicate the presence of biomolecules or the occurrence of physiological alterations4. Therefore, it is possible to hypothesize and test in the classroom simple postulations related to biochemistry and physiological changes such as aging.
Glycogen is a storage carbohydrate, formed by a long and branched chain of glucose molecules formed by glucosyl residues with (1&#8594;4)-&#945; glycosidic linear linkages and (1&#8594;6)-&#945; glycosidic bonds at branch points and is particularly important for muscle contraction, cell differentiation and glycemia maintenance5. Glycogen is synthesized after feeding due to insulin activation of the enzyme glycogen-synthase. During exercise or fasting, epinephrine or glucagon, respectively, activate glycogen phosphorylase and, therefore, break down the polysaccharide to provide glucose-6-phosphate to the muscle cells or release free glucose to circumvent hypoglycemia6,7. Alterations in glycogen levels impacts cell differentiation, signaling, redox regulation, and stemness under various physiological and pathophysiological conditions, including cancer8. In C. elegans, glycogen is mainly found in esophageal muscle, hypodermis, intestine, neurons and mainly in body wall muscles9. The glycogen content can be measured by using Lugol&#39;s Iodine solution, since iodine binds into the helical coils forming an iodine-glycogen complex, giving a visible sharp blue-black or brown-black color, which has been successfully used to demonstrate glycogen content in C. elegans10. It has been demonstrated that glycogen accumulation caused by high glucose feeding can reduce worm&#39;s lifespan, therefore accelerating the aging process11,12. In addition, metabolic disturbances, other hormones, and exposure to xenobiotics can alter glycogen metabolism as well13,14. Therefore, experimentation on glycogen content in C. elegans is quite interesting, since diverse factors may disturb its metabolism and can stimulate an in-class discussion on basic biochemistry associated with transversal themes such as exercise, diets, diseases, and aging.
Aging is a time-dependent functional decline caused by cellular damage. This damage can be associated with oxidative stress, telomere attrition, loss of proteostasis, inflammation and even by accumulation of insoluble polyglucosan bodies15, just to name a few. One of the hallmarks of aging is the reduction of intestinal integrity, associated with several chronic conditions that occur during the life of an organism16. Maintenance of intestinal homeostasis depends on the integrity of the intestinal epithelium, which is supported by junctional proteins forming a physical barrier and connecting adjacent epithelial cells. When there is damage to this epithelium, leakage of luminal content into the interstitium occurs17. Based on this mechanism, the smurf test has been used to verify intestinal integrity in several animal models, since this blue dye Erioglaucine disodium salt does not cross the intestinal membrane, remaining in the lumen18. When worms are infected with a pathogen, contaminated with some toxicants or age, altering the interstitial integrity, the dye crosses the barrier and spreads all over the worm, which becomes all blue. This assay allows discussion on the physiology of aging and experimenting on factors that can accelerate or delay this process by exposing worms to different conditions. The protocols here will describe in detail these two, dye-based methods that can be easily done in class to instigate and stimulate students to formulate and test hypotheses related to biochemistry and physiology.
The first part of protocol shows its applicability to analyze qualitatively and quantitatively the glycogen content in C. elegans model10. The purpose of second part of the protocol is to assess the integrity of the C. elegans intestine. This technique allows for the monitoring of C. elegans aging by evaluating the integrity of the intestinal membranes. Furthermore, it allows evaluating whether a substance accelerates or delays aging and whether any substances have toxic potential on the intestinal barrier19.
The C. elegans strain used for the present study was Bristol N2 wild type. However, the procedure can be replicated using strains that exhibit comparable growth rates, or the method must be adjusted based on need of equipment replacement, considering they have the same or similar function, or depending on the strain used, as certain strains have specific maintenance and/or sensitivity requirements; this information can be obtained from the Caenorhabditis Genetics Center (CGC) or WormBase website. These changes should not impact the reproducibility of the method.

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			<td>37-degree incubator</td>
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			<td>50 mL conical tube</td>
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			<td>6 cm Petri plates&#160;</td>
			<td>Local suppliers</td>
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			<td>Agar bacteriological</td>
			<td>Din&#226;mica Qu&#237;mica Contempor&#226;nea Ltda.</td>
			<td>9002-18-0</td>
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			<td>C. elegans Bristol N2 (wild type)</td>
			<td>Caenorhabditis elegans Genetic Center (CGC, Minnesota, USA)</td>
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			<td>CaCL2</td>
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			<td>10035-04-8</td>
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			<td>Cholesterol</td>
			<td>Sigma-Aldrich Brasil Ltda</td>
			<td>57-88-5</td>
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			<td>D-(+)-Glucose anhydrous&#160;</td>
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			<td>Distilled H2O</td>
			<td>Local suppliers</td>
			<td>-</td>
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			<td>Erioglaucine disodium salt</td>
			<td>Sigma-Aldrich Brasil Ltda</td>
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			<td>Escherichia coli OP50</td>
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			<td>Flow hood</td>
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			<td>Incubator</td>
			<td>Panasonic Healthcare company of North America, MIR-254-PA.</td>
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			<td>KH2PO4</td>
			<td>Din&#226;mica Qu&#237;mica Contempor&#226;nea Ltda.</td>
			<td>7778-77-0</td>
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			<td>Levamisole hydrochloride</td>
			<td>RIPERCOL L 150F</td>
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			<td>Lugol solution</td>
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			<td>S1063-01-AH</td>
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			<td>Microcentrifuge</td>
			<td>Centrifuge 5425R Eppendorf SE, Germany</td>
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			<td>Na2HPO4</td>
			<td>Din&#226;mica Qu&#237;mica Contempor&#226;nea Ltda.</td>
			<td>7558-79-4</td>
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			<td>Din&#226;mica Qu&#237;mica Contempor&#226;nea Ltda.</td>
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			<td>Nystatin</td>
			<td>Sigma-Aldrich Brasil Ltda</td>
			<td>N6261</td>
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			<td>Peptone bacteriological</td>
			<td>&#234;xodo cient&#237;fica</td>
			<td>91079-38-8</td>
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			<td>Stereomicroscope</td>
			<td>Leica S8 Apo Stereomicroscope (S&#227;o Paulo, Brazil)</td>
			<td></td>
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			<td>Streptomycin Sulfate</td>
			<td>Estreptomax</td>
			<td>-</td>
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using low-cost dyes to visualize glycogen accumulation and gut integrity in caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

The glycogen content assay provides a robust and rapid method for screening various testing conditions, such as comparative studies of different strains that may influence glycogen synthesis or degradation. In this study, L4 worms were subjected to three distinct test conditions: fasting, feeding, and glucose-enriched groups. The assay was performed three times, with each condition replicated twice in each assay; a representative image is displayed in Figure 3. Following each assay, worms st...

Read this Scientific Article Protocol about Using Low-cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans at JoVE.com

Author Spotlight: Exploring Metabolic and Aging Processes in C. elegans Using Low-Cost, High-Impact Assays

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

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817 Views.  Federal University of Pampa. Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Video: Author Spotlight: Exploring Metabolic and Aging Processes in C. elegans  Using Low-Cost, High-Impact Assays

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 ...

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, ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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, ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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

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

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...