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 °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 °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 °C for 8 hours.</li>
 <li> Add the desired amount of worms to the plates (if incubated at 37 °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 °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 °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 °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 μl) 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 °C.</li>
 <li>Melted agarose can be kept at 65 °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 μl 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 μl 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/μl.</li>
 <li> Once the ethanol has evaporated completely, add 7 μl 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 μg/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> </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="Bleaching solution effect on fibers; time-lapse microscopy images; recipes 1-5; 3min, 6min duration." data-altupdatedat="1747911167000" data-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 °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="Chilled vs. warm cell culture results; microscopy images; time lapse; 24-hour post-treatment analysis." data-altupdatedat="1747911167000" data-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 °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 °C for 24 hours. Columns on the right display pictures 24 hours later.
<img src="/files/ftp_upload/4019/4019fig3.jpg" alt="Bar chart of treatment effects showing percentages of dead embryos, L1, and adult fragments." data-altupdatedat="1747911167000" data-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 μl 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 °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 μl worm pellet.
<img src="/files/ftp_upload/4019/4019fig4.jpg" alt="Microscopy image and bar chart of organism survival rates at different volumes; analysis of results." data-altupdatedat="1747911167000" data-alt="Figure 4"> 
 Figure 4. Proper aeration is required for hatching and survival of C. elegans embryos. A 100 μl 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 °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='"C. elegans life cycle diagram with developmental stages L1-L4 and adult, microscopy images."' data-altupdatedat="1747911167000" data-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="Microscope images of nematode development stages: L3, L4, Adult; cellular structure analysis." data-altupdatedat="1747911167000" data-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="Fluorescent microscopy image of C. elegans at L3, early L4, late L4, and adult stages; organism growth." data-altupdatedat="1747911167000" data-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="Nematode growth analysis, temperature comparison at 15°C and 25°C, timeline from 16h to 86h, microscopy." data-altupdatedat="1747911167000" data-alt="Figure 8"> 
 Figure 8. C. elegans development at 15 and 25 °C. N2 worms were bleached, incubated overnight in M9 and agitation at 15 °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.

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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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46.5K Views.  Bellvitge Institute for Biomedical Research. Het gemak van het behoud en het uitdragen van de nematode C. elegans Maken het een leuk model organisme om mee te werken. De mogelijkheid synchronisatie wormen kan het werken met een aanzienlijke hoeveelheid onderwerpen aan dezelfde ontwikkelingsfase, wat maakt de studie van een bepaald proces veel dieren.

Video: Basic Caenorhabditis elegans Methods: Synchronization and Observation

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Environment

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

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

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

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

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

Genetics

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

Behavior

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Developmental Biology

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

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

Measurements of Physiological Stress Responses in C. Elegans

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

Measurements of Physiological Stress Responses in C. Elegans

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

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Analyzing Mitochondrial Morphology in C. elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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