Authors would like to acknowledge MICINN (PTA program supporting Montserrat Porta de la Riva), AGAUR (Phd Fellowship to Laura Fontrodona), Instituto de Salud Carlos III (Miguel Servet program supporting Juli&aacute;n Cer&oacute;n), and Marie Curie IRG, ISCIII and IDIBELL for financing the lab.

1. Protocol A: Culturing Worms for Bleaching 11
Large populations of C. elegans can be obtained by culturing them either in liquid media or on solid media in plates. They are usually grown on solid NGM (Nematode Growth Media) and fed with E. coli bacteria, which is added to the plates either alive or dead (killed by UV12, by heat13 or by cold14). The most common procedure uses live OP50 E. coli, which is defective in the synthesis of uracil and cannot overgrow into a thick layer that would obscure the worms.
<ol>
 <li> Mix 3 g of NaCl, 17 g of agar and 2.5 g of peptone and add 975 ml of H2O. Autoclave for 50 min.</li>
 <li> Cool the flask to 55 &deg;C.</li>
 <li> Add 1 ml of 1 M CaCl2, 1 ml of 5 mg/ml cholesterol in ethanol, 1 ml of 1 M MgSO4 and 25 ml of 1 M KPO4 buffer (all of them but cholesterol previously autoclaved).</li>
 <li> Using sterile procedures dispense the NGM solution into Petri plates; fill plates up to 2/3 of their volume.</li>
 <li> Once dry, it is advisable to leave plates at room temperature for 2-3 days before use for detection of contaminants. Prepare a streak of OP50 E. coli from a glycerol stock (OP50 can be obtained from the Caenorhabditis Genetics Center).</li>
 <li> Pick a single colony and grow it in LB overnight at 37 &deg;C with agitation.</li>
 <li> Allow excess of moisture to evaporate from the plates by removing the lid in the laminar flow and add OP50 to the center of the plates with a sterile Pasteur pipette.</li>
 <li> Allow the OP50 E. coli lawn to grow overnight at room temperature or at 37 &deg;C for 8 hours.</li>
 <li> Add the desired amount of worms to the plates (if incubated at 37 &deg;C plates should be cooled at room-temperature before use).</li>
</ol>
TIPS:
 <ul>
 <li>The pouring of the same amount of media in the plates with a pipette or a pump dispenser ensures the same volume of agar to the plates and facilitates the shifting of plate without need to refocus the stereomicroscope.</li>
 <li>Plates (both seeded and unseeded with bacteria) can be used several weeks after prepared when stored in a container at room temperature or 4 &deg;C.</li>
 <li>Avoid plating the bacteria to the edge of the plate. If the lawn extends to the edges of the plate the worms may crawl up the sides, dry out and die.</li>
 <li>Worms live longer if the bacteria seeded on the plates are already dead 15.</li>
</ul>

2. Protocol B: Treatment with Alkaline Hypochlorite Solution ("Bleaching")11
The bleaching technique is used for synchronizing C. elegans cultures at the first larval stage (L1). The principle of the method lies in the fact that worms are sensitive to bleach while the egg shell protects embryos from it. After treatment with alkaline hypochlorite solution, embryos are incubated in liquid media without food, which allows hatching but prevents further development.
<ol>
 <li> Allow worms to grow until adult stage.</li>
 <li> Recover gravid adults in 15 ml tubes by washing plates with M9 buffer.</li>
 <li> Pellet worms by centrifuging for 2 minutes at 400xg (~1500 rpm on a standard table centrifuge) at room-temperature and discard supernatant.</li>
 <li> Perform 1-3 washes until the buffer appears clear of bacteria.</li>
 <li> Add the desired bleaching solution (Table I) and agitate for some minutes (destruction of the adult tissue should be monitored under the dissecting microscope and the reaction stopped when traces of adults are still visible, which typically takes between 3 and 9 minutes depending on several issues, such as the volume of worm pellet, mentioned in the discussion) (Fig. 3).</li>
 <li> Stop reaction by adding M9 buffer to fill the tube.</li>
 <li> Quickly centrifuge (since treatment may still be active) for 1 minute at 400 x g and discard supernatant.</li>
 <li> Wash pellet three more times by filling the tube with M9 buffer.</li>
 <li> Add 1 ml of M9 buffer to the pellet, or place the eggs to unseeded NGM plates, and incubate at the desired temperature with gentle agitation. Proper aeration should be provided (Fig. 4).</li>
</ol>
TIPS:
 <ul>
 <li>There are different bleaching solutions, choose the one that works better in your hands (Table 1, Fig. 1).</li>
 <li>Eggs already laid on the plates can be recovered by scrapping the surface of the agar with a soft material such as a piece of an X-ray film.</li>
 <li>Too many remains of adult animals may impair synchronization as they constitute a food supply for the recently hatched larvae.</li>
 <li>Higher temperatures slightly speed up the development which is inconvenient if any worm skips synchronization because the difference in development between synchronized and unsynchronized worms will be greater at higher temperatures.</li>
 <li>Bleaching solution must be performed just prior to its use. In addition, bleach loses potency after it has been open for a while, in part due to its photosensitivity. We suggest to aliquot each new bottle into small amber bottles to prevent such loss and minimize exposure to light.</li>
 </ul>
3. Protocol C: Worm Plating
<ol>
 <li> Wait between 12 and 24 hours (time to complete embryonic development depends on the temperature) after bleaching was performed and recover worms by centrifugation (2 minutes at 400 x g).</li>
 <li> Discard supernatant, seed worms on the required plates and let remaining liquid dry.</li>
 <li> Place plates at the required temperature.</li>
</ol>
TIPS:
 <ul>
 <li> L1 larvae in M9 buffer can be kept at 15 &deg;C rocking at least for one week without obvious alterations.</li>
 <li> be careful when calculating the worms you will seed because too many may exhaust the food faster than expected and ruin your experiment. Approximately 500 L1 can reach adulthood in a 55 mm plate without running out of food. </li>
 </ul>
4. Protocol D: C. elegans Observation
D.1 Nomarski observation 
Differential interference contrast microscopy is an optical microscopy illumination technique used to enhance the contrast in unstained transparent samples. The word Nomarski refers to the prism used, named after his inventor. By observing animals alive we are able to examine the physiology of the animal with the only alterations derived from immobilization. In addition, as no fixative is added, fluorescent markers can be observed in vivo. This fact and the possibility of fusing fluorescent markers to a gene of interest make it feasible to follow processes in which the protein of study may be involved. By using the technique described in this protocol, not only live worms can be observed, but they can also be recovered and plated again.
Agar pad preparation (just before use):
<ol>
 <li> Prepare agarose 2% in water and melt. Keep melted at 65 &deg;C.</li>
 <li> Place two slides with a piece of tape on them at both sides of a third, clean slide.</li>
 <li> Using a Pasteur pipette place a drop of agar onto the clean surface.</li>
 <li> Cover the agar with another clean slide placed on top of the three slides perpendicularly.</li>
 <li> Press gently so the agar drop is flattened to the thickness of the tape spacers.</li>
 <li> Once the agar solidifies, gently pull out the taped slides and separate the two remaining slides by sliding one relative to the other.</li>
</ol>
Mounting live animals
<ol start="7">
 <li> Place one drop (10 &mu;l) of levamisole 1mM or sodium azide 10-30 mM onto the center of the pad.</li>
 <li> Transfer animals into the drop using a worm pick.</li>
 <li> Gently place a coverslip over the animals and fix it at both sides with some nail polish or silicone.</li>
</ol>
TIPS:
 <ul>
 <li>Keep aliquots of agarose 2% at 4 &deg;C.</li>
 <li>Melted agarose can be kept at 65 &deg;C for at least one day.</li>
 <li>Note: Levamisole is a nicotinic receptor agonist which elicits spastic muscle paralysis16.</li>
 <li>Be cautious, Sodium Azide is extremely toxic!</li>
 </ul>
D.2 Ethanol fixation and DAPI staining
The protocol described here represents a fast way of dyeing worms with DAPI, however because of the dissecation of the worm some structures may present some alteration. There are several other methods to fix worms previous to DAPI staining such as fixation with Carnoy's solution or formaldehyde that preserve better the integrity of the worm 17.
Ethanol fixation (modified from 18)
<ol>
 <li> Place ~10 &mu;l of M9 buffer (or water) on a microscope slide.</li>
 <li> Using a worm pick carefully transfer 10-25 worms to the drop.</li>
 <li> Using filter paper or a micro-pipette remove as much M9 buffer as possible without removing the worms or letting them dry.</li>
 <li> Add ~10 &mu;l of 90% ethanol and let it dry.</li>
 <li> Repeat step 4 once or twice.</li>
</ol>
4',6-diamidino-2-phenylindole (DAPI) staining
<ol start="6">
 <li> Mix DAPI with the desired mounting media to a final concentration of 2 ng/&mu;l.</li>
 <li> Once the ethanol has evaporated completely, add 7 &mu;l of the DAPI:mounting media mixture.</li>
 <li> Place a coverslip and fix it at both sides with some nail polish or silicone. Slides will be ready for observation approximately 5 min after the addition of DAPI.</li>
</ol>
TIPS:
 <ul>
 <li>The mounting media contains glycerol, so a small amount is enough to cover the whole preparation.</li>
 <li>There exists a wide variety of commercial mounting media (Fluoromount or Prolong, for example), their quality and price depend on how long you want to store your sample.</li>
 <li>Be careful, DAPI is a known mutagen which binds strongly to A-T rich regions in DNA.</li>
</ul>
Recipes
Nematode Growth Medium (NGM)
1.7% (w/v) Agar 
50 mM NaCl 
0.25% (w/v) Peptone 
1 mM CaCl2 
5 &mu;g/ml Cholesterol 
25 mM KPO4 
1 mM MgSO4 
M9 buffer
22mM KH2PO4 
42 mM Na2HPO4 
86 mM NaCl 
1 mM MgSO4
Bleaching solutions tested 
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>recipe #1</td>
 <td>recipe #2</td>
 <td>recipe #3</td>
 <td>recipe #4</td>
 <td>recipe #5</td>
 </tr>
 <tr>
 <td>water (ml)</td>
 <td>2.75</td>
 <td>3.5</td>
 <td>0.5</td>
 <td>0.5</td>
 <td>1.5</td>
 </tr>
 <tr>
 <td>sodium hydroxide (ml)</td>
 <td>1.25 (1M)</td>
 <td>0.5 (5M)</td>
 <td>2.5 (1M)</td>
 <td>2.5 (2M)</td>
 <td>2.5 (1M)</td>
 </tr>
 <tr>
 <td>sodium hypochlorite ~ 4% (ml)</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 <td>2</td>
 <td>1</td>
 </tr>
 <tr>
 <td>total (ml)</td>
 <td>5</td>
 <td>5</td>
 <td>4</td>
 <td>5</td>
 <td>5</td>
 </tr>
 </tbody>
</table>
Table I. Different bleaching solution recipes tested for this article. Recipes #3 and #4 are 2x, and should be added to the same volume of M9. Recipes for #1, #2 and #5 have been previously reported 2, 11, 19. Final concentrations: #1 NaOH 0.25M, NaOCl ~0.8%, #2 NaOH 0.5M, NaOCl ~0.8%, #3 NaOH 0.625M, NaOCl ~1%, #4 NaOH 1 M, NaOCl ~1.6%, #5 NaOH 0.5M, NaOCl ~0.8%.



5. Representative results
<img src="/files/ftp_upload/4019/4019fig1.jpg" alt="Figure 1" /> 
 Figure 1. Comparison of five different bleaching solutions at two different incubation times. N2 worms washed twice with M9 were split into five 15 ml conical tubes containing each bleaching solution. Tubes were shaken vigorously and 1 ml transferred to a new tube with M9 to stop the reaction after the time specified. After bleaching procedure worms were incubated with 1 ml of M9 at 20 &deg;C for 24 hours. In each case, lower picture was taken just after bleaching, upper picture 24 hours later.
<img src="/files/ftp_upload/4019/4019fig2.jpg" alt="Figure 2" /> 
 Figure 2. Temperature of bleaching solution affects the effectiveness of the treatment. Equal volumes of N2 worms were bleached with the same bleaching solution either previously chilled on ice for 20 minutes or kept at 25 &deg;C for the same time. The two columns on the left show pictures just after bleaching. After treatment worms were incubated in 15 ml conical tubes with 1 ml of M9 at 20 &deg;C for 24 hours. Columns on the right display pictures 24 hours later.
<img src="/files/ftp_upload/4019/4019fig3.jpg" alt="Figure 3" /> 
 Figure 3. The ratio worm pellet:time of alkaline hypochlorite incubation affects the effectiveness of the treatment. 50, 100, 250 and 500 &mu;l of worm pellet were incubated with 2 ml of bleaching solution #3 for 3, 6 and 9 minutes. Hatched L1, dead embryos and remains of adult fragments were quantified after incubation at 20 &deg;C for 24 hours in 15 ml conical tubes with 1 ml of M9 buffer. Approximately three confluent 55 mm plates with adult worms are needed to get a 100 &mu;l worm pellet.
<img src="/files/ftp_upload/4019/4019fig4.jpg" alt="Figure 4" /> 
 Figure 4. Proper aeration is required for hatching and survival of C. elegans embryos. A 100 &mu;l pellet of N2 worms were bleached for 6 minutes and incubated in 15 ml conical tubes with 1, 5 or 10 ml, as specified, of M9 at 20 &deg;C for 24 hours. The upper part of the figure displays pictures of the cultures after 24 hours, where arrows indicate eggs that did not hatch. At the bottom, there is a graph depicting the amount of larvae (light grey) and dead embryos (dark grey) 48 hours after bleaching at the stated conditions.
<img src="/files/ftp_upload/4019/4019fig5.jpg" alt="Figure 5" /> 
 Figure 5. Life cycle of C. elegans. a . Approximate length of the worms at different stages. Hours required to reach each stage depending on the temperature (modified from 20). b. Nomarski (up) and DAPI (down) pictures of different worms at the indicated developmental stages. Most significant features in each phase are magnified. L1: arrow indicates the precursors of the somatic gonad and the germ line. Early L4: black arrow (Nomarski) indicates the developing vulva; white arrows (DAPI) indicate the two gonadal arms. Mid-late L4: arrow indicates the developing vulva at the so-called Christmas tree stage. Young Adult: black arrow indicates an embryo inside the uterus, arrowhead points to the spermatheca, white arrow indicates an oocyte. Gravid adult: arrowhead (DAPI) points out fertilized embryos. Arrow in DAPI image indicates spermatheca.

<img src="/files/ftp_upload/4019/4019fig6.jpg" alt="Figure 6" /> 
 Figure 6. Vulva morphology at L3, L4 and Adult stages. At the L3 stage only a small lumen where the vulva is formed can be observed. At L4, this lumen expands forming the so-called "Christmas tree". In the adult the vulva is already closed. Yellow lines indicate the location of the vulva at these three stages.
<img src="/files/ftp_upload/4019/4019fig7.jpg" alt="Figure 7" /> 
 Figure 7. DAPI staining at L3, early L4, late L4 and Adult stages. At L3, germ line is elongated. At L4, gonad arms present U-shape morphology. At late-L4 stage sperm can be observed in the distal part of the gonad. Young Adults present oocytes. The Adult germ line presents oocytes and embryos. Yellow lines delimitate germ lines at the different stated stages.
<img src="/files/ftp_upload/4019/4019fig8.jpg" alt="Figure 8" /> 
 Figure 8. C. elegans development at 15 and 25 &deg;C. N2 worms were bleached, incubated overnight in M9 and agitation at 15 &deg;C, transferred to plates and grown the indicated times at the stated temperatures.

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We have nothing to disclose.

Nematode Synchronization
Several bleaching solutions have been described. We tried five different recipes (Table I) and, in our hands, they did not show significant differences in the synchronization of worm populations (Fig. 1). However, our experiments did show that parameters such as temperature (Fig. 2), the ratio bleaching solution:volume of worms (Fig. 3) and the volume of M9 with which the embryos are incubated for hatchin...

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.

Watch this Scientific Journal Video about Basic Caenorhabditis elegans Methods: Synchronization and Observation at JoVE.com

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.

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 ...

basic-caenorhabditis-elegans-methods-synchronization-and-observation

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46.1K Views.  Bellvitge Institute for Biomedical Research. 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.

Video: Basic Caenorhabditis elegans Methods: Synchronization and Observation

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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

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

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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, ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

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 ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

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

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...