Name: a high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of caenorhabditis elegans
Uploaded: 2019-03-14T14:45:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans at JoVE.com

The authors thank CGC for kindly sending the C. elegans. This work was supported by National Key Research and Development Program of China (#2018YFC1603102, #2018YFC1602705); National Natural Science Foundation of China Grant (#31401025, #81273108, #81641184), The Capital Health Research and Development of Special Project in Beijing (#2011-1013-03), the Opening Fund of the Beijing Key Laboratory of Environmental Toxicology (#2015HJDL03), and the Natural Science Foundation of Shandong Province, China (ZR2017BF041).

The protocol follows the animal care guidelines of the Animal Ethics Committee of the Beijing Center for Disease Prevention and Control in China.
1. Chemical preparation
<ol>
	<li>Obtain chemicals (Table 1 and Table of Materials).</li>
	<li>Determine the highest and lowest dosage of the individual chemicals for a minimum concentration of 100% lethality (LC100, 24 h) and a maximum concentration of 100% nonlethality (LC0, 24 h) to worms. Use at least six dilutions of the highest concentration (Table 1). 
	NOTE: Conduct a preliminary worm lethality test9 to explore LC100 and LC0 for a new chemical, to determine the dosage.</li>
	<li>Dilute each chemical with K-medium (Table of Materials) to 2x the required concentration. Use K-medium as a control to compare the phenotype alterations caused by the chemicals.
	<ol>
		<li>For example, prepare 7 gradient concentrations of cadmium chloride (CdCl2) (Table 1). To prepare 2x the highest concentrated aqueous solution (4.64 mg/mL), dissolve 92.8 mg of CdCl2 solid powder in 8 mL of K-medium and fill up to 10 mL after the powder has fully dissolved. Prepare the other concentration levels by dilution with K-medium.</li>
	</ol>
	</li>
	<li>Prepare eight parallel wells for every concentration in the chemical gradient. Each well contains 50 &#181;L of the 2x chemical solution. Prepare at least three groups of eight parallel wells of K-medium as&#160;controls (Table 2). 
	NOTE: In brief, a volume of 500 &#181;L of 2x working solution is necessary for a single dose of each chemical.</li>
</ol>
2. Worm preparation
<ol>
	<li>Obtain wild-type N2 worms and Escherichia coli OP50 strains from the Caenorhabditis Genetics Center (CGC).</li>
	<li>Obtain synchronized L4 worms.
	<ol>
		<li>Pick a single colony of E. coli OP50 from the streak plate. Aseptically inoculate the colony in 100 mL of LB broth and grow it overnight at 37 &#176;C. 
		NOTE: The E. coli OP50 solution is now ready for seeding to nematode growth medium (NGM, Table of Materials) plates.</li>
		<li>Pour NGM into a 90 mm plastic Petri plate. Seed each plate with 300 &#181;L of E. coli OP50 solution the day after pouring. Incubate N2 worms on the NGM plates with OP50 at 20 &#176;C for about 2-3 days until most of the worms have reached the adult stage.</li>
		<li>Harvest gravid worms into a 15 mL sterile conical centrifuge tube with sterile H2O. Let the worms settle down for at least 2 min, aspirate the H2O, and add 5 mL of bleach buffer (Table of Materials).</li>
		<li>Vortex the tube for 5 min, spin the tube for 30 s (at 1,300 x g) to pellet the eggs, and discard the supernatant.</li>
		<li>Wash the eggs with 5 mL of sterile H2O and vortex the tube for 5 s. Centrifuge the tube for 30 s (at 1,300 x g), remove the supernatant, and wash again.</li>
		<li>Pipette the eggs onto a new NGM plate with OP50. Incubate them at 20 &#176;C. Monitor the hatched L1 worms the next morning; the worms will reach the L4 stage in approximately 40 h.</li>
	</ol>
	</li>
	<li>Wash the L4 worms off the 90 mm Petri plates with K-medium into a 50 mL sterile conical tube. Adjust the concentration of worms to ~40 animals per 100 &#181;L of K-medium under a stereomicroscope. Add 50 &#181;L (~20 worms) into each well of the 384-well plate. These synchronized worms (L4 stage) are ready for the following treatment by chemicals.</li>
</ol>
3. Chemical treatment and video capture
NOTE: In a 384-well plate, worms (50 &#181;L in each well) are treated to six to seven dosages of an individual chemical (Table 1). Prepare eight parallel wells, each containing 50 &#181;L of the 2x chemical solution for every dosage (eight wells are filled with the same chemical and the same concentration, Table 2). All videos are collected using a digital camera attached to an inverted microscope (Table of Materials). The chemical treatment experiment lasts for 24 h. Do not add bacterial food to each well during the 24 h chemical treatment experiment.
<ol>
	<li>Before adding the chemicals, set the 384-well plate with the synchronized worms on the automatic stage and take videos of each well with the programmed acquisition procedure (7 frames per second for 2 s; it takes ~25&#160;min to scan each plate).</li>
	<li>Add 50 &#181;L of the 2x chemical stock prepared according to section 1 for each well (Table 2). Set the time as the 0 h point.</li>
	<li>Incubate the 384-well plate at 20 &#176;C and shake it at 80 rpm in an incubator shaker.</li>
	<li>Remove the plate from the incubator and transfer it to an automatic stage. Take videos of each well of the whole plate, at 12 h and at 24 h, to check the phenotypes of the worms for each specific chemical treatment in K-medium. Approximately 25 min are required for one plate screen.</li>
</ol>
4. Experiment video processing
NOTE: A program for experimental video and images processing was written and packaged. It can be freely downloaded (see Table of Materials). The experimental video is stored in the form of an image frame sequence, and the frame sequence of each video is stored in a specific directory. The program can recognize worms and quantify phenotypes automatically.
<ol>
	<li>In the graphical user interface (GUI, Figure 1), add the parameters, such as the frame sequence directory, the output directory, the worm size parameter, and the movement threshold parameter. Click the Analyze button to process the experimental images.
	<ol>
		<li>Click the Select button to choose the source images directory.</li>
		<li>Add the middle result directory in the interface. 
		NOTE: The middle results include the segmented images. These middle results are useful for the visual observation of the processed images.</li>
		<li>Add the final result directory in the interface.</li>
		<li>Add the average worm size parameter in the Worm Size textbox in the interface. 
		NOTE: The size parameter used in the experiments is 2,000.</li>
		<li>Add the Threshold of moved ratio in the interface. 
		NOTE: The ratio used in the experiments is 0.93.</li>
		<li>Click the Analyze button to start the image processing. Click the Reset button to clear the added parameters. 
		NOTE: There are 33 features defined and quantified for worms. All the defined phenotypes are sorted by categories (listed in Table 3). These features can be quantified from experimental images. A quantitative comparison among different chemicals, which have different toxicities, can be done by comparing these features.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The advantages of C. elegans have led to its increasing usage in toxicology9, both for mechanistic studies and high-throughput screening approaches. An increased role for C. elegans in complementing other model systems in toxicological research has been remarkable in recent years, especially for the rapid toxicity assessment of new chemicals. This article provides a new assay of high-throughput, quantitative screening of worm phenotypes in a 384-well plate for the automatic ident...

Along with the rapid development of chemical compounds applied to industrial production and people&#39;s daily life, it is important to study the toxicity testing models for the chemicals. In many cases, the rodent animal model is employed to evaluate the potential toxicity of different chemicals on health. In general, the determination of lethal concentrations (i.e., the assayed 50% lethal dose&#160;[LD50] of different chemicals) is used as the traditional parameter in a rodent (rat/mouse) model in vivo, which is time-consuming and very expensive. In addition, due to the reduce, refine, or replace (3R) principle that is central to animal welfare and ethics, new methods that allow for the replacement of higher animals are valuable to scientific research1,2,3. C. elegans is a free-living nematode that has been isolated from soil. It has been widely used as a research organism in the laboratory because of its beneficial characteristics, such as a short lifespan, easy cultivation, and efficient reproduction. In addition, many fundamental biological pathways, including basic physiological processes and stress responses in C. elegans, are conserved in higher mammals4,5,6,7,8. In a couple of comparisons we and others have made, there is a good concordance between C. elegans toxicity and toxicity observed in rodents9. All of this makes C. elegans a good model to test the effects of chemical toxicities in vivo.
Recently, some studies quantified the phenotypic features of C. elegans. The features can be used to analyze the toxicities of chemicals2,3,10 and the aging of worms11. We also developed a method that combines a liquid worm culturing system and an image analysis system, in which the worms are cultured in a 384-well plate under different chemical treatments12. This quantitative technique has been developed to automatically analyze the 33 parameters of C. elegans after 12-24h of chemical treatment in a 384-well plate with liquid medium. An automated microscope stage is used for experimental video acquisition. The videos are processed by a custom-designed program, and 33 features related to the worms&#39; moving behavior are quantified. The method is used to quantify the worm phenotypes under the treatment of 10 compounds. The results show that different toxicities can alter the phenotypes of C. elegans. These quantified phenotypes can be used to identify and predict the acute toxicity of different chemical compounds. The overall goal of this method is to facilitate the observation and phenotypic quantification of experiments with C. elegans in a liquid culture. This method is useful for the application of C. elegans in chemical toxicity evaluations and phenotype quantifications, which help predict the acute toxicity of different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.&#160;In addition, this method can be applied to the toxicity screening and testing of new chemicals or the compound as the food additive agent pollution, pharmacautical compounds, environmental exogenous compound, and so on.

<table><tbody><tr><td>2-Propanol</td><td>Sigma-Aldrich</td><td>59300</td><td></td></tr><tr><td>384-well plates</td><td>Throme</td><td>142761</td><td></td></tr><tr><td>Agar</td><td>Bacto</td><td>214010</td><td></td></tr><tr><td>Atropine sulfate</td><td>Sigma-Aldrich</td><td>PHL80892</td><td></td></tr><tr><td>Bleach buffer</td><td></td><td></td><td>0.5 mL of 10 M NaOH, 0.5 mL of5% NaClO, 9 mL ofultrapure water</td></tr><tr><td>Cadmium chloride</td><td>Sigma-Aldrich</td><td>202908</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>21074</td><td></td></tr><tr><td>CCD camera</td><td>Zeiss</td><td>AxioCam HRm</td><td>Zeiss microscopy GmbH</td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>Copper(II) sulfate</td><td>Sigma-Aldrich</td><td>451657</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>24105</td><td></td></tr><tr><td>Ethylene glycol</td><td>Sigma-Aldrich</td><td>324558</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>K-Medium</td><td></td><td></td><td>3.04 g of NaCl and 2.39 g of KCl in 1 L ultrapure water</td></tr><tr><td>LB Broth&amp;nbsp;</td><td></td><td></td><td>10&amp;nbsp;g/L&amp;nbsp;Tryptone,&amp;nbsp;5&amp;nbsp;g/L&amp;nbsp;Yeast&amp;nbsp;Extract,&amp;nbsp;5&amp;nbsp;g/L&amp;nbsp;NaCl&amp;nbsp;</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>NGM Plate</td><td></td><td></td><td>3 g ofNaCl, 17 g ofagar, 2.5 g ofpeptone in 1 L of ultrapure water, after autoclave add 1 mL of cholesterol (5 mg/mL in ethanol), 1 mL of MgSO4 (1 M), 1 mL of CaCl2 (1 M), 25 mL of PPB buffer</td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>60130</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma-Aldrich</td><td>795496</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>795488</td><td></td></tr><tr><td>PPB buffer</td><td></td><td></td><td>35.6 g of K2HPO4, 108.3 g of KH2PO4 in 1 L ultrapure water</td></tr><tr><td>shaker</td><td>ZHICHENG</td><td>ZWY-200D</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>71382</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma-Aldrich</td><td>s7920</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>71690</td><td></td></tr><tr><td>Sodium hypochlorite solution</td><td>Sigma-Aldrich</td><td>239305</td><td></td></tr><tr><td>The link of program</td><td></td><td></td><td>https://github.com/weiyangc/ImageProcessForWellPlate</td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1625</td><td></td></tr><tr><td>Zeiss automatic microscope&amp;nbsp;</td><td>Zeiss</td><td>AXIO Observer.Z1</td><td>Zeiss automatic microsco with peproprietary software Zen2012 and charge coupled device(CCD) camera</td></tr></tbody></table>

a high-throughput assay for the prediction of chemical toxicity by automated phenotypic profiling of caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

We have tested the phenotypes of worms exposed to different concentrations of more than 10 chemicals12. In the test, 33 distinct features were quantified for each chemical compound at three time points (0 h, 12 h, and 24 h). Previously, a comparison between a manual and an automatic analysis of a lifespan assay was done11,12. In this assay, we found that chemicals and concentrations can influence the worm p...

Watch this Scientific Journal Video about A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans at JoVE.com

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

a-high-throughput-assay-for-prediction-chemical-toxicity-automated

Research

JoVE Journal

Environment

7.2K Views.  Beijing Center for Disease Prevention and Control/Beijing Center of Preventive Medicine Research, China. C.elegans toxicologische test is waardevol. Dit protocol beschrijft hoe c.elegans te behandelen met chemicaliën in een 384-put plaat, video's vast te leggen en toxicologische gerelateerde fenotypes te kwantificeren. Deze methode kan helpen bij het identificeren en voorspellen van de potentiële acute toxiciteit van een chemische stof.Deze kwantitatieve techniek is ontwikkeld om automatisch de 33 parameters van C.elegans te analyseren na 12 tot 24 uur chemische behandeling in een 384-p...

Video: A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Developmental Biology

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biology

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Immunology and Infection

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicine

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Neuroscience

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

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 ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

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