This study was supported by the Deutsche Forschungsgemeinschaft (DFG) within the IRTG 1874 &#34;Diabetic microvascular complications&#34; and CRC 1118 &#34;Reactive metabolites as a cause for diabetic late complications&#34;. C. elegans strains N2 and CL2166 were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

NOTE: This protocol is divided into five sections. In sections 1&#8211;3, the main protocol to culture C. elegans at a large-scale is presented. Sections 4 and 5 provide additional protocols for the assessment of exemplified metabolites occurring in diabetic metabolites. In detail, section 1 describes a general large-scale culture on plates. Section 2 focuses on the transfer of large amounts of C. elegans, whereas section 3 explains the harvesting of a large-scale sample. Section 4 explains the protein isolation from the sample and section 5 describes the sample preparation for subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses.
1. Large-scale Culture on Plates
<ol>
	<li>In general, work under a sterile hood or next to an open flame to avoid contaminations.</li>
	<li>To culture worms until they reach the young adult stage, follow this previously published protocol7 with the following adaptations.
	<ol>
		<li>Use living Escherichia coli OP50 bacteria to feed the nematodes and prepare 400 &#181;M fluorodeoxyuridine agar (not ampicillin/FUdR) for experiments.</li>
		<li>For the preparation of a synchronous population, use unconcentrated OP50, and for succeeding experiments with the adults, use 3.3x concentrated OP50 by removing 70% of the supernatant.</li>
		<li>Daily feeding intervals are recommended. When assessing oxidative stress, different intervals and volumes for feeding might interfere with the results.</li>
		<li>For inoculation, take a plate with several nematodes and cut it into pieces of approximately 0.5 x 1 cm2 with a scalpel. Transfer two to three pieces onto nematode growth media (NGM) plates of 60 mm in diameter, containing unconcentrated OP50.</li>
		<li>Incubate the plates at the temperature suitable for the strain (wild-type N2 should be cultured at 20 &#176;C) until eggs are visible on the plate.</li>
		<li>Wash off most of the eggs and adult animals&#160;with 2 mL of M9 buffer and collect them in a 15 mL tube. Centrifuge the suspension at 800 x g for 2 min. Meanwhile, start preparing the bleaching solution.</li>
		<li>Take out the centrifuged tube and remove the M9 buffer with a 10 mL pipet. Add 10 mL of the bleaching solution and mix on the vortex until the adult nematodes are lysed. This takes usually approx. 5&#8211;7 min, but should not take longer than 15 min or the eggs will not develop fully later on in the experiment.</li>
		<li>Centrifuge the suspension at 800 x g for 2 min. Wash 3 times with 10 mL M9 buffer to clear the eggs from bleaching solution.</li>
		<li>Now add 150 &#181;L of the egg suspension on OP50 plates. A density of 200&#8211;300 eggs should be reached on each plate. Wait until nematodes reached the adult stage. 
		NOTE: The M9 buffer (pH 7.0) used for the depicted experiments consists of the following substances: 22 mM KH2PO4, 42 mM Na2HPO4, and 86 mM NaCl. After autoclaving the mixture, add 1 mM MgSO4. The bleaching solution contains 0.5 M NaOH, 2.3 mM NaOCl solved in distilled water.</li>
		<li>Prepare sterile M9 buffer, sterile pipette tips with a cut tip to wash off the worms, and 15 mL tubes to collect them in.</li>
		<li>Apply approximately 1 mL of M9 buffer on the plates,&#160;gently distribute the buffer by swinging and gently pipet&#160;the nematodes off the plate.</li>
		<li>Now, collect the nematodes in the tube. Apply 150 &#181;L of suspension directly on the plates (seeded with OP50) with the cut pipette tips, and evaluate microscopically the density of the nematodes (recommended: 50&#8211;100 nematodes per 60 mm plate).</li>
		<li>If they are too dense, dilute the suspension with M9 buffer and evaluate it under the microscope until the desired density is reached. If the yield is too little, let the worms settle down or centrifuge the suspension 10 s at 800 x g to remove fluid. 
		NOTE: An example of a sufficient density is given in Figure 1A (macroscopic) and 1B (microscopic) at 20X augmentation. Proceed empirically as described.</li>
		<li>Keep in mind that the worms settle down quickly. To ensure an equal distribution between the plates, invert the tube after every stack of plates (4&#8211;5 plates). 
		NOTE: The cut pipette tip, as well as the gentle swinging and washing of the plates, reduces shear stress.</li>
	</ol>
	</li>
</ol>
2. Transfer of Large Amounts of Caenorhabditis elegans
<ol>
	<li>Wash off the nematodes with approximately 500 &#181;L of M9 buffer, depending on the age and dryness of the plates. Using the same buffer, repeat detaching the nematodes until most of the animals are collected in&#160;suspension.</li>
	<li>Slowly take up the nematodes with a cut pipette tip and transfer them on a plate prepared with OP50. Let the plate dry. 
	NOTE: Be careful not to apply much more than the recommended volume. Especially in long-term experiments, the plates might not tolerate it and the agar can break, allowing the nematodes to crawl into the cracks.</li>
</ol>
3. Harvest of a Large Caenorhabditis elegans Sample
<ol>
	<li>Depending on the selected method, start experiments with a sufficient amount of C. elegans. For LC-MS/MS analyses, prepare 20 plates (60 mm x 15 mm) per group with approximately 100 nematodes per plate to ensure a yield of at least 200 &#181;g of protein per sample. 
	NOTE:&#160;This part of the protocol does not require working under sterile conditions anymore, as the nematodes will be collected and frozen.&#160;</li>
	<li>On the day of the sample collection, prepare liquid nitrogen to snap-freeze the collected samples. Keep non-sterile M9 buffer on ice to slow down the nematode&#8217;s metabolism.</li>
	<li>Wash off the C. elegans from the plates by applying 1 mL of M9 buffer and gently swirling the plates. This avoids collecting deceased nematodes and most eggs, if there are any present, because they stick to the bacteria and agar.</li>
	<li>Take up the volume and transfer it to a 15 mL tube.</li>
	<li>After the collection of the complete sample, wait for the nematodes to settle or centrifuge the tube briefly at 800 x g and remove most of the M9 buffer. Wash the sample 3x with approximately 10 mL of M9 (10x the sample volume) to remove any bacteria. 
	NOTE: To ensure that all deceased nematodes are removed, sucrose floatation is another option. However, this was not suitable for the displayed experiments15. Sucrose is hydrolyzed to glucose and fructose. In the depicted experiments, the role of glucose was assessed to promote biochemical changes found in chronic diabetes. Thus, sucrose floatation could potentially confound the results.</li>
	<li>Prepare one 1.5 mL tube with 1 mL of M9 buffer and one empty 1.5 mL tube for every sample. Transfer the pellet with a glass pipet&#160;from the 15 mL tube to the empty 1.5 mL tube. This step is crucial to ensure a quantitative transfer. 
	NOTE: Unlike during previous steps, here the nematodes are more concentrated. Because of a possible adherence to plastic pipet&#160;tips, loss of a large part of the sample is expected. Therefore, use glass pipettes instead.</li>
	<li>After the transfer, use the glass pipette to take up 1 mL of M9 buffer from the second 1.5 mL tube to rinse the nematodes from the pipette wall. Also, wash the remaining nematodes from the 15 mL tube and transfer them to the sample tube.</li>
	<li>Centrifuge the sample tube at 19,000 x g for 1 min and then remove as much supernatant as possible.</li>
	<li>Seal the tube with Parafilm and immediately snap-freeze it in liquid nitrogen. Store the tube at -80 &#176;C until the lysate preparation.</li>
</ol>
4. Protein Isolation for Mass Spectrometry
NOTE: The protein isolation described here can also be used for other assays (e.g., western blot).
<ol>
	<li>To the frozen sample, add the same volumes of 0.5 mm zirconium oxide&#160;beads and at least 2x the volume&#160;of lysate buffer containing a proteinase inhibitor cocktail. 
	NOTE: The depicted nematodes-to-protein correlation analyses were performed using 100 &#181;L of buffer. LC-MS/MS samples were lysed with 200 &#181;L of buffer.</li>
	<li>Start the homogenizer for 1 min on speed 9. Ensure that the samples are completely lysed. Repeat this step if they are not fully lysed.</li>
	<li>Centrifuge the samples for 30 min at 4 &#176;C at 19,000 x g. Transfer the supernatant to a new tube on ice and store it at -80 &#176;C until further measurements are going to be taken. 
	NOTE: The non-denaturing buffer used for the depicted experiments consists of the following substances: 20 mM Tris HCl (pH 8), 137 mM NaCl, 1% Triton-X, and 2 mM EDTA. 
	NOTE: Having followed the steps of this protocol and having applied the suggested scale, the yield of a protein pellet from approximately 1,000 nematodes should be approximately 500 &#181;g. For more comparisons, consult Figure 2 and the Representative Results.</li>
</ol>
5. Sample Preparation for Liquid Chromatography-tandem Mass Spectrometry Measurements
NOTE: Depending on the parameter of interest, the sample preparation will differ. This protocol focuses on methylglyoxal and AGE determination.
<ol>
	<li>Measure intracellular methylglyoxal by derivatization with 1,2-diaminobenzene (DB) as previously described9.
	<ol>
		<li>Homogenize the samples with 20 &#181;L of ice-cold 20% (wt/vol) trichloroacetic acid in 0.9% (wt/vol) sodium chloride and 80 &#181;L of water in a 1.5 mL tube.</li>
		<li>Spike the samples of 5 &#181;L with the internal standard (13C3-MG; 400 nM), mix them by vortexing, and then centrifuge them at 21,000 x g for 5 min at 4 &#176;C.</li>
		<li>Transfer 35 &#181;L of the supernatant to the HPLC sample vial containing a 200 &#181;L glass insert.</li>
		<li>Add 5 &#181;L of 3% sodium azide (wt/vol) to each sample followed by 10 &#181;L of 0.5 mM DB in 200 mM HCl containing 0.5 mM diethylenetriaminepentaacetic acid (DETAPAC) in water.</li>
		<li>Incubate the samples for 4 h at room temperature, protected from light.</li>
		<li>Analyze the samples by LC-MS/MS, as previously described9. 
		NOTE: For the measurement of AGEs, isolate the proteins as described in section 4 of the protocol.</li>
	</ol>
	</li>
	<li>Measure the protein concentration of the (thawed) lysates using a Bradford assay10.
	<ol>
		<li>Prepare aliquots of 30 &#181;L for a standard curve spiked with bovine serum albumin (BSA) solved in phosphate-buffered saline (PBS) of the following concentration: 0; 0.1; 0.2; 0.3; 0.4; 0.6; 0.8; and 1.0 mg/dL. 
		NOTE: If the sample was prepared at the suggested size (see step 4.1 for the preparation of lysate with 200 &#181;L of buffer), the estimated concentration should be in the range of approximately 2&#8211;7 mg/mL. In this case, dilute the samples 1:10 with PBS prior to the measurement. 
		NOTE: For the first experiments using this method, it is recommended to measure the samples in different dilutions (1, 1:10, and 1:100). As there is no exact determination of the number of nematodes, the yield can differ between individual researchers.</li>
		<li>Use a transparent 96-well plate (polystyrene) and pipette 10 &#181;L of each standard concentration and of the samples in the chosen dilutions in duplicates in the wells.</li>
		<li>Dilute the protein assay dye reagent concentrate 1:5 with distilled water (aq. dest.) and add 200 &#181;L of the dilution to every sample on the plate. Incubate the plate for 10 min at room temperature.</li>
		<li>Measure the absorbance within 30 min at 595 nm with a plate reader. The samples can be interpolated from the calculated standard curve. More details of the method have been described extensively in the literature10.</li>
	</ol>
	</li>
	<li>Measure the intracellular AGEs as previously described11,12.
	<ol>
		<li>Wash the total protein extracts (approximately 200 &#181;g) 3x with water by ultracentrifugation in 10 kDa centrifugal filter units.</li>
		<li>Add 10 &#181;L of 100 mM HCl, 10 &#181;L of pepsin (2 mg/mL in 20 mM HCl), and 10 &#181;L of thymol (2 mg/mL in 20 mM HCl) to the recovered protein (approximately 100 &#181;L) and incubate the samples at 37 &#176;C for 24 h.</li>
		<li>Neutralize and buffer the samples at pH 7.4 by adding 12.5 &#181;L of 0.5 M potassium phosphate buffer (pH 7.4) and 5 &#181;L of 260 mM KOH.</li>
		<li>Add 10 &#181;L of Pronase E [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)] and 10 &#181;L of a penicillin-streptomycin solution, and incubate the samples at 37 &#176;C for 24 h.</li>
		<li>Add 10 &#181;L of aminopeptidase [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)] and 10 &#181;L of prolidase [2 mg/mL in 10 mM potassium phosphate buffer (pH 7.4)], and incubate the samples at 37 &#176;C for 48 h.</li>
		<li>Analyze the resulting hydrolysate by LC-MS/MS, as previously described12.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

This protocol presents a reliable approach for the large-scale culturing of C. elegans to obtain quantitative results. Findings from the literature could be replicated as shown in the Representative Results. Even though this protocol for the collection of large-scale samples of C. elegans seems like a straight-forward method, there are certain pitfalls to take into account. Regarding the synchronization of the nematode population, this protocol describes an approach by bleaching the pop...

Protein analyses, such as a western blot or mass spectrometry, require milligrams of protein. This yield requires a large-scale culturing of hundreds of C. elegans, which can be accomplished either by liquid culture or on solid media transferring the nematodes by washing. Fluid and shear stress induces the expression of epithelial sodium channels (ENaC), which could increase the osmotic stress through an increased uptake of sodium, potentially altering the lifespan of C. elegans and affecting metabolic analyses1. Therefore, some critical steps in this protocol for the plate-based approach take the reduction of stress affecting experimental variability into account. Liquid culture, on the other hand, influences the phenotype of the nematodes and complicates the culture and collection of an exact number of nematodes2. Moreover, reactive substances can be altered by media components and may distribute unevenly before reaching the nematodes. Regarding the limitations of liquid culture, this protocol provides an alternative approach to culturing large-scale samples of C. elegans.
C. elegans is a model organism with a distinct network of 302 neuronal cells, making up one-third of all its cells3. Since its introduction into science, many homologous and orthologous genes have been described, amplifying its value as a model for medical research. Recently, evidence for neurological impairment in diabetic retinopathy, preceding vascular damage, has been presented4. C. elegans is lacking blood vessels, but contains a distinct neuronal network, making it a suitable model to investigate neuronal alterations apart from vascular ones. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. Biochemical alterations in diabetic complications involve the formation of AGEs, which further influence the formation of ROS in response to hyperglycemia5. AGEs are found in C. elegans and contribute to neuronal damage6. Chronic diseases are often caused by complex, polygenic processes requiring a multiparametric approach for the assessment of their underlying mechanisms, as exemplified here with the assessment of diabetic complications. This protocol can be of use for obtaining multiple parameters simultaneously, as well as subsequently. Increased comparability and reproducibility of a multiparametric approach can be achieved by omitting the morphological and metabolic differences between liquid and solid media culture.

<table><tbody><tr><td>&lt;em&gt;E. coli &lt;/em&gt;OP50</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>&lt;em&gt;C. elegans &lt;/em&gt;N2</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>&lt;em&gt;C. elegans &lt;/em&gt;CL2166</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>Petri dish, 60 mm x 15 mm</td><td>Greiner One</td><td>628161</td><td></td></tr><tr><td>Volumetric pipet, glas, 10 mL</td><td>Neolab</td><td>E-0413</td><td></td></tr><tr><td>Proteinase inhibitor cocktail tablets</td><td>Roche</td><td>04693124001</td><td></td></tr><tr><td>&lt;u&gt;Non-denaturing lysate buffer:&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris-HCl, pH 8</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Sodiumchloride (NaCl)</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X-100</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>E5391</td><td></td></tr><tr><td>96-well plates, transparent bottom</td><td>Brand</td><td>781611</td><td></td></tr><tr><td>Infinite M200, plate reader</td><td>Tecan</td><td>30017581</td><td></td></tr><tr><td>Zirconium Oxide Beads, 0.5 mm</td><td>Next advance</td><td>ZROB05-RNA</td><td></td></tr><tr><td>Bullet Blender, homogenizer</td><td>Next advance</td><td>BBX24</td><td></td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma</td><td>P6887</td><td></td></tr><tr><td>Thymol</td><td>Sigma</td><td>T0501</td><td></td></tr><tr><td>Pronase E/ Protease from &lt;em&gt;Streptomyces griseus &lt;/em&gt;</td><td>Sigma</td><td>P6911</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Sigma</td><td>P43339</td><td></td></tr><tr><td>Prolidase from Porcine Kidney</td><td>Sigma</td><td>P6675</td><td></td></tr><tr><td>Aminopeptidase from&lt;em&gt; Aeromonas proteolytica&lt;/em&gt;</td><td>Sigma</td><td>A8200</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Merckmillipore</td><td>UFC501096</td><td></td></tr><tr><td>Basic Materials for plate culture are described in Reference &lt;sup class=&quot;xref&quot;&gt;6&lt;/sup&gt;.</td><td></td><td></td><td></td></tr></tbody></table>

plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Here examples of creating a large-scale C. elegans culture for applications in diabetes research are presented. It can be of interest to relate the parameters to a single animal, rather than to normalize it to the total protein concentration. In an assay requiring a small number of nematodes, this can be easily accomplished by counting the nematodes. For a large-scale C. elegans culture involving hundreds of nematodes per experimental group, this approach is inconvenient...

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Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

plate-based-large-scale-cultivation-caenorhabditis-elegans-sample

Research

JoVE Journal

Biochemistry

6.3K Views.  Heidelberg University. Dit protocol beschrijft een methode voor de grootschalige teelt van Caenorhabditis elegans op solide media. Als alternatief voor vloeibare cultuur kunnen dit protocol verkrijgen van parameters voor verschillende schalen onder plaat gebaseerde teelt. Dit verhoogt de vergelijkbaarheid van de resultaten door het weglaten van de morfologische en metabole verschillen tussen vloeibare en vaste mediacultuur.

Video: Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Neuroscience

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Immunology and Infection

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Biology

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

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

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

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

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

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

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

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

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

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

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

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