We thank Dipa Bhaumik, Aaron Miller and Bob Hughes for technical assistance. Strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We are grateful to Georgia Woods and members of the Lithgow, Kapahi and Melov Labs for useful discussions. M.L. was supported by National Institutes of Health (NIH) Training Grant T32 AG000266 and an Ellison Medical Foundation/American Federation of Aging Research Post-Doctoral Fellowship. This work was supported by a grant from BioAge Labs, a Larry L. Hillblom Foundation grant, as well as National Institutes of Health grants UL1024917, supporting the Interdisciplinary Research Consortium on Geroscience, and 1R01AG029631-01A1 to G.J.L.

1. Preparing Buffers
<ol>
	<li>LB broth
	<ol>
		<li>Use pre-mixed granules (see Materials Table) and follow manufacturer&#39;s protocol for preparation.</li>
	</ol>
	</li>
	<li>Nematode Growth Media (NGM) Agar
	<ol>
		<li>Mix 23 g of agar, 3 g of NaCl, 2.5 g of bacto peptone, and deionized water to 0.972 L. Autoclave and let cool to 60 &#176;C, then add 25 mL of 1 M potassium phosphate (KPO4) buffer (pH 6.0), 1 mL of 1 M magnesium sulfate (MgSO4), 1 mL of 1 M calcium chloride (CaCl2), and 1 mL of 5 mg/mL cholesterol (in ethanol).</li>
	</ol>
	</li>
	<li>Hypochlorite solution
	<ol>
		<li>Mix 5 mL of 5 M KOH, 4 mL of sodium hypochlorite solution (6%), and deionized H2O to a total volume of 50 mL.</li>
	</ol>
	</li>
	<li>S basal
	<ol>
		<li>Mix 5 g of NaCl, 1 g of K2HPO4, 6 g of KH2PO4, and de-ionized water to 1 L, then autoclave.</li>
	</ol>
	</li>
</ol>
2. Preparation of Concentrated E. coli (OP50)
NOTE: This step concentrates 1 L of a saturated E. coli solution into 25 mL of S basal buffer.
<ol>
	<li>Inoculate 5 mL of autoclaved LB broth (step 1.1) with a single colony of E. coli (Strain OP50), and incubate for 4-6 h at 37 &#176;C with shaking (250 rpm). Use 0.1 mL of this solution to inoculate 1 L of LB in a 2 L Erlenmeyer flask. Incubate the 1 L flasks overnight (12-16 h) at 37 &#176;C with shaking (250 rpm).</li>
	<li>Centrifuge 1 L overnight cultures in 500 mL centrifuge bottles for 5 min at 10,000 x g and 4 &#176;C to pellet the bacteria and decant away remaining media. Resuspend pellet in 25 mL of S Basal (step 1.4). Store at 4 &#176;C.</li>
</ol>
3. Preparation of NGM Culture Plates
<ol>
	<li>For mass culture plates used to produce large numbers of worms, dispense 30 mL of molten (60 &#176;C) NGM agar (step 1.2) in a laminar flow cabinet onto 10 cm culture plates with an automated fluid dispensing apparatus or a serological pipette. Allow to dry overnight.</li>
	<li>Pipet 2 mL of concentrated OP50 onto each 10 cm plate, spread to completely cover the agar surface by tilting and rotating the plate, then allow plates to dry. Store plates at 4 &#176;C for up to 2 weeks.</li>
	<li>For high throughput assay culture plates used for screening, utilize an automated 8 channel dispenser to dispense 0.15 mL of molten NGM agar into each well of a 96 well plate in a laminar flow cabinet. Use care to make sure wells are devoid of bubbles, as these will enable burrowing of worms that will compromise the assay in that well. Allow to dry overnight. Store plates at 4 &#176;C for up to 2 weeks.</li>
</ol>
4. Preparation of Temperature Sensitive (ts) Sterile C. Elegans Mass Cultures
<ol>
	<li>To start cultures, using a &#39;worm pick&#39; (a glass pipette with an attached platinum wire or a manufactured worm pick) under a dissecting microscope, transfer 20 eggs (strain TJ1060: spe-9(hc88)I; rrf-3(b26)II) onto mass culture plate(s) prepared in step 3.1. Then, incubate plates at 20 &#176;C for 6 days (allows for more than one generation of growth, as you are targeting for collection of the progeny of the 20 eggs moved).</li>
	<li>Visually confirm presence of gravid adults (eggs present in the uterus) with a dissecting microscope. Collect the gravid adults by dispensing 2-3 mL of S basal onto the plate using a glass pipette. Swirl the liquid around the plate by hand, then collect the liquid into a 15 mL tube using a glass pipette.</li>
	<li>Spin down the tube (1,000 x g for 1.5 min at 20 &#176;C) to pellet worms, then aspirate off supernatant. Add 10 mL hypochlorite solution (step 1.3) to the worm pellet and rapidly shake the tube by hand in a top-to-bottom direction for 5 s. Incubate for 5 min, shaking every 2.5 min.</li>
	<li>Again, spin down the tube (1,000 x g for 1.5 min); the pellet should be yellowish-brown. Aspirate off the supernatant. Add 10 mL hypochlorite solution to the egg pellet and vigorously shake the tube. Incubate 1-3 min, with occasional shaking, and while monitoring appearance with the dissecting microscope. Visually confirm only eggs are remaining, and proceed to the next step.</li>
	<li>Spin down the tube (1,000 x g for 1.5 min) and aspirate off the supernatant. The pellet should be white with no brown coloration. 
	NOTE: After the removal of the hypochlorite solution, it is critical to use sterile techniques and buffers, since contamination (bacterial and fungal, in particular) can ruin the experiment, wasting valuable time and reagents. This study used a filtered laminar flow cabinet and moved liquids using only sterile pipettes and tips.</li>
	<li>Open the tubes in a sterile space. Wash the pellet 3 times with S basal, spinning down (1,000 x g for 1.5 min) and aspirating away the supernatant each time. Re-suspend the egg pellet in 2-3 mL S basal. These eggs can now be used for an overnight hatch in buffer to collect L1 larva (step 5).</li>
</ol>
5. Hatching C. Elegans Eggs in Buffer to Get Synchronous Cultures (Preparing L1 Larval C. Elegans )
<ol>
	<li>Decant the egg pellet and buffer into a sterile glass petri dish with a cover. Rinse the remaining eggs from the tube into a dish using a serological pipette with 2-3 mL of S basal. Add 5 mL of S basal to ease crowding, as this may encourage hatching, as will light shaking (20 rpm with a belly dancer orbital shaker). Incubate overnight at 20 &#176;C, to allow eggs time to hatch (16-24 h). All hatchlings will arrest at the 1st larval stage (L1) due to the absence of food. 
	NOTE: Anecdotal evidence indicates that fresh L1 preparations (16-36 h after egg collection) generate the most synchronous populations of adult worms.</li>
	<li>Collect L1s in a 15 mL tube with a serological pipette. Spin down L1s in a centrifuge (1,000 x g for 1.5 min), aspirate away the supernatant, and repeat until all L1s have been collected from hatching plates, while leaving 10 mL of S basal for next step.</li>
	<li>From the tube containing L1s, micropipette out 0.01 mL (immediately after mixing) and dispense it onto an unseeded (no E. coli) NGM plate. Count the number of L1s on the plate. Repeat 10 times and obtain an average for the number of L1s in each aliquot. Generally, expect between 1-3 L1s per &#181;L (when starting with 1 mass culture plate containing 20 eggs).</li>
	<li>Determine with a serological pipette the volume of S basal holding the L1s. Then extrapolate with the determined average number of worms per 0.01 mL, obtained in the previous step, to estimate the total number of L1s. Generally, expect between 10,000-30,000 L1s (per mass culture plate containing 20 eggs).</li>
	<li>To use this culture in 96-well assay, dilute the L1 suspension to 1 L1 per &#181;L in a sterile round media bottle, then proceed to step 7. If this culture is to be used to generate additional C. elegans mass cultures, then proceed to step 6.</li>
</ol>
6. Large Scale Synchronous Culture Preparation
NOTE: L1 solutions can be used to quickly increase worm populations.
<ol>
	<li>To generate large worm numbers with an L1 solution, dispense up to 5,000 L1s on each of the desired number of mass culture plates. Collect gravid adults 2.5 days later (20 &#176;C) as described in steps 4.1-2, and proceed to step 4.3 to collect the eggs.</li>
	<li>As it has been anecdotally observed that repeated hypochlorite collection can stress the subjugated population, split populations by picking eggs for propagation prior to subjecting the remaining population to hypochlorite treatments, so that multiple generations of the population are not repeatedly subjected to hypochlorite solution treatment.</li>
</ol>
7. Setup of 96-Well Assay Plates
<ol>
	<li>Allow the assay plates (prepared in step 3.3) to reach room temperature. Then, in a laminar flow cabinet, add 5 &#181;L of concentrated OP50 (prepared in step 2.2) to each well. As before, use an automated 8 channel dispenser.</li>
	<li>Add 10 &#181;L of L1 suspension to each well using an automated 8 channel dispenser. This will yield on average 10 worms per well, since they are present in the suspension at 1 L1 per &#181;L. To promote equal distribution of L1s from the slurry, dispense L1s from a round media bottle actively mixed with a moderately slowly turning stir bar.</li>
	<li>Cover plate with lid, box batches of plates, and incubate at 25 &#176;C for 2 days.</li>
</ol>
8. Treating 1 st Day Adult C. elegans with Chemicals
<ol>
	<li>Remove chemical library plates from the freezer, and allow plates to reach room temperature before proceeding. 
	NOTE: Chemical library plates as described here are 96 well plates containing discrete compounds in each well, all of which are at the same concentration and dissolved in DMSO. These libraries do not utilize the outer columns, and so each plate has a maximum of 80 distinct chemicals. In the protocol described here, the library concentration is 10 mM. It is important to consider solvent effects on phenotypes. While this study screens at a DMSO concentration of 0.5%, this level was never exceeded in these lifespan assays and in low throughput assays, where it is more likely to pick a compound stock solution concentration. This protocol does not exceed 0.25%, which is a level at which lifespan modifications do not occur16, at least in the N2 strain.</li>
	<li>Gently shake plate at 60 rpm on a microplate shaker for 1 min immediately prior to use.</li>
	<li>Using an automated 96-well liquid handler, transfer 0.75 &#181;L of compound (or DMSO) from the library plate to the assay plate. The depth of the pipette during liquid delivery to the assay plate is controlled carefully, such that the pipette tips of the liquid handler are delivering the 0.75 &#181;L volume to the ~15 &#181;L of liquid on the surface of the agar (with worms and concentrated OP50 from steps 7.1-2) and does not pierce the agar surface in the well. Do not use manual 8 or 12 multi-channel pipettes except in very small screens (&#60;10 plates), as they can increase error and often result in puncturing of the agar surface, which compromises the assay.</li>
	<li>Prepare chemical library negative control plates with DMSO (dimethyl sulfoxide), assuming the library being tested uses DMSO as the solvent. Make 1 negative control plate for every 5 test plates, up to 10 negative control plates. If possible, also make a positive control plate (NP11 at 20 mM, for a final assay concentration of 0.1 mM, is effective for this purpose; see Representative Results section). Do not add chemicals (test or control) to the 2 exterior columns of the plates, as they are particularly susceptible to desiccation.</li>
</ol>
9. Drying Cultures
<ol>
	<li>To remove liquid from the surface of the agar, dry plates in the laminar flow cabinet for 4-8 h. It is critical that these plates are dried completely but do not become desiccated. As some plates will take longer to dry than others, monitor all plates closely and remove as soon as they are dry. Confirm drying by careful visual inspection of the individual wells of the plate. 
	NOTE: Depending on factors that include the exhaust rate of the cabinet, the time this takes must be empirically determined. This drying step can take about 4-8 h for a full cabinet.</li>
	<li>Seal plates with transparent film, then put on the plate lids.</li>
	<li>Spin the 96-well assay plates for 45 s at 1,000 x g.</li>
	<li>Store plates inverted at 25 &#176;C.</li>
</ol>
10. Scoring C. Elegans Cultures for Longevity
<ol>
	<li>Monitor negative control plates until &#62;95% mortality is observed. Check plates weekly for the first two weeks and daily thereafter. Caenorhabditis cohorts of the same strain and species can display significantly different lifespans by trial17. 
	NOTE: For the lifespan assay described here, with TJ1060 animals cultured at 25 &#176;C, 95% mortality in these populations has occurred from adult day 16-20.</li>
	<li>When negative control wells are considered to have reached the &#62;95% mark, spin down all 96-well assay plates for 45 s on a table top centrifuge with a microplate adapted rotor (250 x g for 45 s at 20 &#176;C).</li>
	<li>Score all wells in the control plates. This is done by counting and recording the number of alive and dead animals in each well. Dead worms can be differentiated from alive worms by their complete lack of movement. Provoke movement in alive worms by dropping the 96 well plate from approximately 7 cm onto the dissecting microscope surface while looking through the eyepiece for any evoked response. Dead worms will not respond, and nearby lab mates may not appreciate the repetitive noise.</li>
	<li>For the test plates, count and record only wells containing live worms. When a live worm is observed, count all alive and dead animals in that well, along with the well position and plate. 
	NOTE: It is often useful to re-score the plates after &#62;99% of negative control worms are dead. In large scale screens, this may be the best point to do the initial scoring. In either case, score/re-score as described above</li>
</ol>
11. Re-Testing Compounds
<ol>
	<li>When testing only a small number of candidate compounds (&#60;320 compounds), as with a re-test or a candidate screen (see Representative Results), restrict the assay to the 32 central wells in an effort to minimize possible plate effects. This leaves the 2 wells closest to the edge of the plate untreated but still containing agar, food, and worms.</li>
</ol>

<ol>
	<li>Lucanic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+activation+of+a+food+deprivation+signal+extends+lifespan.">Chemical activation of a food deprivation signal extends lifespan.</a> Aging Cell. , (2016).</li><li>Evason, K., Huang, C., Yamben, I., Covey, D. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+that+confer+thermal+stress+resistance+and+extended+lifespan.">Compounds that confer thermal stress resistance and extended lifespan.</a> Exp Gerontol. 43 (10), 882-891 (2008).</li><li>Hansen, M., Hsu, A. L., Dillin, A., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Genes+Tied+to+Endocrine,+Metabolic,+and+Dietary+Regulation+of+Lifespan+from+a+Caenorhabditis+elegans+Genomic+RNAi+Screen.">New Genes Tied to Endocrine, Metabolic, and Dietary Regulation of Lifespan from a Caenorhabditis elegans Genomic RNAi Screen.</a> PLoS Genet. 1 (1), 17 (2005).</li><li>Lee, S. 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J., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+dietary+restriction+for+genetic+epistasis+analysis+and+gene+discovery+in+C.+elegans.">Optimizing dietary restriction for genetic epistasis analysis and gene discovery in C. elegans.</a> PLoS One. 4 (2), 4535 (2009).</li><li>Hamilton, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19 (13), 1544-1555 (2005).</li><li>Frankowski, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethyl+sulfoxide+and+dimethyl+formamide+increase+lifespan+of+C.+elegans+in+liquid.">Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid.</a> Mech Ageing Dev. 134 (3-4), 69-78 (2013).</li><li>Lucanic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+genetic+background+and+experimental+reproducibility+on+identifying+chemical+compounds+with+robust+longevity+effects.">Impact of genetic background and experimental reproducibility on identifying chemical compounds with robust longevity effects.</a> Nat Commun. 8, 14256 (2017).</li></ol>

The authors declare that no funding entity supporting this work had any input on collection of data, analysis of results, or the decision to publish.

Here we have described a simple method for culturing C. elegans in 96-well plates. We describe these cultures for use in chemical screening and lifespan assays, but they can be used for many types of assays. While multi-well culture conditions for chemical screening have previously been reported8, including for lifespan analysis10, the assay described here differs in several ways. The lifespan assay described here utilizes 96-well plates, relies on sterile mutants (instead of chemical sterilization), uses agar culture conditions, uses simple liquid handling to move worms, and is manually scored at the endpoint. The different approaches used in this assay may be of help to researchers looking for screening methods that include these conditions.
Crucial elements to this protocol mainly center on the quality of the plates produced for the assay. First, when making plates it is imperative that the agar surface is free of bubbles and other imperfections. These variations in the agar surface almost always lead to burrowing and loss of the well. Second, this protocol relies on drying off liquids deposited during the setup and drugging stages. It is critical that these liquids are completely removed, but the agar does not become dried out. Anecdotally, it appears that worms in wells that are not completely dried (swimming in liquid) live longer than properly dried wells, while over-drying of the plates leads to desiccation and cracking of the agar, which leads to worm burrowing and loss. Another crucial element of this protocol is maintaining sterile conditions. As with any lifespan assay, there is a lot of work involved with the plate setup, and many opportunities and time exist for assay ruining contamination to settle and grow on the plates.
While the lifespan assay described here is useful for screening of compounds in C. elegans, it is limited to particular genetic backgrounds, as it relies on a temperature sensitive (ts) sterile strain; we used TJ1060, which has high sterility penetrance. This restricts the strains available for screening with this method. Alternative approaches including crossing ts sterile mutations into the desired background or using chemical sterilization. We have not tried chemical sterilization with this assay, but it should be possible. Potential hurdles include placing the sterilizer on the worms at the correct stage and in the proper dose. The protocol described here uses a liquid dispenser that is fast, but does not appear to dispense adults effectively. So, these worms develop on the plates and therefore must be treated with the sterilization chemical on the plates. Determining the optimal dose and timing would be important for success. Alternative strategies include using a dispensing method that can move adults. In the past, we have found that machines able to identify and sort adults are generally much slower than the simple liquid handlers, which can dispense homogenous slurries of suspended larva.
In general, we use this method to rapidly screen chemicals and treatments for large positive effects on lifespan. The method is particularly effective when used with multiple doses, high replicate numbers, and close monitoring of negative controls that are always maintained and interspersed among the test plates. Positive controls are also useful when designing and implementing the screens described in this manuscript. However, to be an effective positive, the control would need to be fairly potent and/or highly robust. When the screen was originally deployed, a suitably potent and robust compound could not be identified. Currently, there are many reports in the &#39;aging&#39; literature of likely candidates for positive controls useful in the screens described here. As described in the candidate screen section of the expected results section of this manuscript, and in Figure 2 and Table 1, the compound NP1 is an effective positive control for this screen. We generally confirm positive hits from these screens with standard lifespan assays on 3 mm culture plates.

Here a protocol is described that was developed for high throughput screening of chemical compounds for effects on Caenorhabditis elegans lifespan. The protocol itself is readily adaptable to other phenotypic screens, including studies utilizing reporter C. elegans strains. This protocol was successfully utilized to identify a novel biologically active compound, NP1 (nitrophenyl-piperazine 1), that strongly extends the lifespan of C. elegans through a dietary restriction mechanism. NP1 and a characterization of its effect on lifespan, including a description of the genetic pathways at play, was previously described elsewhere1. That report also includes a description of the results from a large-scale implementation of the screen. Here we describe in much greater detail the methodology and setup of the screen itself, which is scalable for both small- and large-scale applications.
The goal that led to the creation of the protocol described here was to develop a C. elegans culture platform that allowed for the rapid screening of large chemical libraries for novel biologically active compounds. While chemical screens have been performed prior to the development of this protocol, these were mainly small-scale and/or candidate type approaches2,3. Indeed, most of the screens performed in the lab on tens of compounds utilized stress resistance assays, with hits being screened for longevity effects4. This study sought instead to directly screen for lifespan effects. Remarkably, there were few descriptions in the scientific literature at the time of performing large scale chemical screens with C. elegans. Indeed, compared to other types of screens5,6,7, large-scale chemical screening using C. elegans seemed to be under-employed.
There were two descriptions of large scale chemical screens in C. elegans that were used as a basis to develop this approach. The first of these was an elegant study from Peter Roy&#39;s laboratory using chemical screens to identify compounds that could perturb the development of animals. The study then followed up with a forward mutagenesis screen to identify the genetic targets of these chemicals8. The protocol described in that study used 24-well pates, which was simply unworkable for this study&#39;s specific needs (limited amounts of chemical in the library and limited available incubator space). The other study was Michael Petrascheck&#39;s innovative and ambitious small-molecule screen for lifespan extending chemicals9,10. That study used 384-well plates and liquid cultures. The other main feature of this screen was the use of FUDR (5-fluorodeoxyuridine) to chemically inhibit progeny production. After considerable consideration of the protocol described in that study, both chemical sterilization and liquid cultures of C. elegans were avoided.
Although FUDR is widely used in C. elegans lifespan experiments, there were concerns that FUDR might cause unanticipated drug interactions, or that FUDR itself may have some effect on lifespan. Recently this latter concern has been validated, at least at some concentrations and in particular at 25 &#176;C11. To circumvent the problem of progeny contamination, C. elegans strain TJ1060 was used, which harbors two independent temperature sensitive mutations (spe-9(hc88)I; rrf-3(b26)II) that abolish sperm production and have been demonstrated to be useful for mass cultures of synchronous populations12. This strain was completely sterile at 25 &#176;C, but will produce progeny when cultured at 15-20 &#176;C. This study used conventional culture conditions, with worms crawling on the surface of agar, as results obtained with liquid cultures are not always reproducible with conventional agar plates. While this phenomenon is not completely understood, it has been demonstrated that worms cultured in liquid respond to DR (dietary restriction) in a different manner than worms cultured on standard media13,14. It is therefore plausible that liquid media could mask some DR mimetics that would otherwise be identified with the screen.
Having settled on agar culture conditions, different options for scoring the performance of wells in this assay were considered. While various automated or imaging based assays were available, they necessitated acquisition and deployment of technically challenging devices, most of which were prohibitively expensive. Whilst considering these options, it was essential to refer to previous lifespan screens of all types. Ultimately, this study used a scoring method adapted from the Siu Sylvia Lee Lab&#39;s whole genome RNAi screen for lifespan phenotypes15. Specifically, all lifespan plates were set up in the same manner, but only the negative controls were monitored. Once the negative control plates were confirmed to have near complete mortality, test plates were scored by hand. In this large-scale screen, positives were confirmed by re-testing with freshly prepared chemicals, using triplicate repeats of the primary positives.

<table><tbody><tr><td>LB Broth Miller Granules</td><td>VWR</td><td></td><td></td></tr><tr><td>Unispense&amp;nbsp;</td><td>Wheaton Science Products</td><td></td><td>automated fluid dispensing apparatus</td></tr><tr><td>96 well assay plate; COSTAR 3370</td><td>Corning</td><td></td><td></td></tr><tr><td>Multidrop 384; automated 8 channel dispenser</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>manufactored worm pick</td><td>Genesee Scientific</td><td></td><td></td></tr><tr><td>Belly Dancer Orbital Shaker</td><td>Stovall Life Science inc.</td><td></td><td></td></tr><tr><td>microplate shaker; MTS 2/4 Digital</td><td>IKA Works, inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>automated 96-well liquid handler; Biomek FX Liquid Handler</td><td>&amp;nbsp;Beckman Coulter</td><td></td><td></td></tr><tr><td>automated 96-well liquid handler; Bench Top Pipettor&amp;nbsp;</td><td>Sorenson Bioscience, inc.</td><td></td><td></td></tr><tr><td>manual 8 or 12 multi-channel pipettes</td><td>we use rainin&amp;hellip;</td><td></td><td></td></tr><tr><td>transparent 96 well film cover; TempPlate RT Optical Film&amp;nbsp;</td><td>USA Scientific</td><td></td><td></td></tr><tr><td>table top centrifuge; Centrifuge 5810 R</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

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.

We begin by examining representative results from employing this protocol for 96-well lifespan assays. In the first example, the method was used to screen through 30,000 chemicals to successfully identify chemicals that extend the lifespan of C. elegans, and also includes results from the follow-up re-test screen of the best performing chemicals using the same protocol. These results were previously described1. The second example uses the same protocol in a smaller-scale screen of candidate compounds.
Large-scale screen and re-test: The large-scale screen testing 30,000 compounds was performed at a single dose, in duplicate. Each chemical was therefore tested in two wells with an expected total animal population of 20. To accomplish this, three discrete sets of 10,000 compounds (in duplicate) were screened over a period of ~3 months to cover the entire 30,000 compounds. This was done in part, to ensure a manageable amount of manual scoring at the end of the assay and required a total of 260 of the 96-well assay plates in each of the sets. In library stock plates, only 80 wells contained compound; one replicate of 10,000 compounds makes 125 assay plates. This study used 2 replicates with 10 negative control plates in each set. To increase throughput, plates were scored when negative control populations were assessed to have experienced ~99% mortality. All test wells were then examined for live worms.
Chemicals in wells with one live worm were called hits. Chemicals in wells containing more than 1 worm alive were also called hits, and additionally all the worms in those wells (alive and dead) were counted to generate a simple score (percent alive score; the number of alive worms divided by the number of dead worms). Each chemical in the library could therefore be called a hit twice and those hits could, but might not, have scores associated with them. 512 distinct chemicals were called hits (at least once) from the screen of 30,000 compounds. The screen was performed with the intention of identifying potent and/or robust (reproducible) pro-longevity effects. These two different properties may have distinct effects. Potent chemicals could dramatically increase lifespan, in which case we would expect a high percent of worms to be alive in that chemical&#39;s well. For robust chemicals, both replicates would be expected to have live worms. The hit list was ranked based on these criteria. 179 of the chemicals yielded high percent alive scores, or were hit in both replicates and were therefore chosen for re-testing. Most of the remaining chemicals that were called hits, but not selected for re-testing, had only contained a single worm alive in one of the replicate wells.
Re-testing of the hits was performed, similar to the primary screen except with 3 replicates (expected total animal population of 30), and careful quantification of the lifespans of the animals in the untreated control plates. These changes were added to aid in comparing the test and control wells. 179 of the chemical hits were re-ordered, diluted to 10 mM, and moved into 96 well plates. Only the inner 24 wells were used for the re-test assay (current versions of this protocol would have used the inner 32 wells as described in the protocol section for re-test screens) and the compounds were re-tested in triplicate with six negative control assay plates treated with DMSO solvent. One negative control plate was scored periodically for total survival (Figure 1A). When approximately 95% of control animals were dead, all plates were completely scored (all treated live and dead worms were counted).
To call hits from the re-test results, a cut-off was made at the average percent alive score of the negative control +2 standard deviations (SD). For the test wells, averaged percent alive (at the time of scoring) scores were calculated by averaging the percent alive score from three replicates for each well. For this particular assay, negative control wells&#39; average percent alive scores were calculated as the average of three replicates for 48 wells (2 sets of triplicate negative control plates). The SD used to call hits from the re-test chemical set was the SD across the 48 negative control averaged percent alive scores. Using this strategy, this study found 57 hits from the re-test library of 179 compounds, demonstrating that a total of 32% of the re-test chemicals tested positive (Figure 1B). Binning of the averaged percent alive scores for the negative control and test wells indicated that the test wells outperformed the control wells (Figure 1C).
Candidate screen: Small scale screens can be performed in a manner similar to the retest described above. Here, we describe the results from a candidate screen with 139 compounds. These candidates were assembled as structures likely to promote longevity. For this screen, we performed 5 replicates of the test plates at two different doses (50 &#181;M and 100 &#181;M). Additionally, we used 8 negative control plates, with a single positive control (NP1) plate containing two different doses; 50 &#181;M and 100 &#181;M.
Negative control plates were monitored until ~90% of animals were dead. At that time, wells were scored by counting live and dead worms in all control wells, and from test wells that contained live worms. The percent alive at the time of scoring were used to calculate averaged percent alive scores for each well. For the negative controls, 32 wells had percent alive scores from 8 replicates. For the positive controls, there were 4 well scores averaged across 4 replicates for each dose. Hits were called for wells with an averaged percent alive score that was greater than the averaged negative control percent alive score +2 SD. This cutoff left 14 hits from the candidate screen and excluded all of the negative control wells. That cutoff also included all of the positive control wells treated at 100 &#181;M and 50% of the positive controls treated at 50 &#181;M. These results are presented here as binned averaged percent alive scores for both the 50 &#181;M (Figure 2A) and the 100 &#181;M (Figure 2B) test screens.
Finally, all plates were re-scored at a late time point, which corresponded to a time when greater than 99% of the negative control treated animals were dead. Those results indicated that the positive control wells far out-performed the negative control and test wells (Figure 2C). While the test wells were slightly better than the negative controls. This latter result is biased by the indication that many of the test wells appeared to exhibit toxicity relative to the control treatment (Table 1), thereby confounding this simple interpretation. This was to be expected, since the candidate library was a true screen of chemicals with unknown biological activity in C. elegans, while the re-test screen was essentially pre-screened for compounds that were not toxic. Since this primary screen was for compounds that lengthened lifespan, significantly toxic chemicals should not have been in the re-test set.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56892/56892fig1.jpg" /> 
Figure 1: Representative results from a high throughput screen and re-testing 
(A) Survivorship curve from a representative negative control plate used in the re-test assay. This curve was constructed from scoring all live and dead worms in the 24 wells used in this assay 4 times over the 18 days of the assay. At day 18 of adulthood, controls were assessed to have ~5% survival and all control test and plates were then also scored. (B) Table summarizing the results of the screen and re-test. (C) Binning of the wells averaged percent alive scores for both control and test conditions from the re-test screen. For controls, scores are from 48 wells each consisting of the average of 3 replicates. Test results are from 179 wells each consisting of the averaged score from three replicates. Figure 1 is reproduced from a previous publication1.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56892/56892fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56892/56892fig2.jpg" /> 
Figure 2: Representative results from a candidate screen 
(A) Binning of the results from the 50 &#181;M candidate screen. The 139 test-well results represent the averaged percent alive score from 5 replicates. The 32 negative control results consist of averaged percent alive scores from 8 replicates. The 4 positive control results consist of averaged percent alive scores from 4 replicates (16 total wells containing NP1 at 50 &#181;M). (B) Binning of the results from the 100 &#181;M candidate screen. All is the same as in (A), except that the test wells and positive controls were treated at 100 &#181;M concentrations. The same negative control plates are shown in both (A and B). (C) Representative results from re-scoring the plates at a relatively extreme late time point. Positive controls dramatically outperformed all others, if just accounting for the percent of wells with live worms at this time point, which is biased against test wells as described in main text and Table 1.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56892/56892fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="6">Binning of Averaged Percent Alive Scores&#160;</td>
		</tr>
		<tr>
			<td>Grouped Scores</td>
			<td>All Dead (0)</td>
			<td>1-10</td>
			<td>11-20</td>
			<td>21-30</td>
			<td>31-40</td>
			<td>&#62;41</td>
		</tr>
		<tr>
			<td>Re-test&#160;</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td># of (-) Control</td>
			<td>16</td>
			<td>31</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td># of Test Wells (50&#181;M)</td>
			<td>46</td>
			<td>69</td>
			<td>50</td>
			<td>8</td>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Candidate Screen</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td># of (-) Control</td>
			<td>0</td>
			<td>11</td>
			<td>21</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td># of (+) Control (50&#181;M)</td>
			<td>0</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td># of (+) Control (100&#181;M)</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td># of Test Wells (50&#181;M)</td>
			<td>94</td>
			<td>22</td>
			<td>21</td>
			<td>2</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td># of Test Wells (100&#181;M)</td>
			<td>86</td>
			<td>25</td>
			<td>21</td>
			<td>6</td>
			<td>1</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Representative results from binning different screens 
Here we present the binning of the averaged percent alive scores from the two screens (re-test and candidate). In the re-test screen, the peak is similar to the negative control with the re-tests outperforming the negative control dramatically on the right (long-lived) side of the table. This indicates the presence of multiple pro-longevity compounds present in the re-test set. In the candidate screen, we see that the peak is in the all dead category, while there is also an apparent signal from the right side of the table. This indicates the presence of pro-longevity compounds in the candidate set, but also indicates the presence of significant toxicity among the candidate set of compounds.

Watch this Scientific Journal Video about A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans at JoVE.com

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.

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 ...

a-simple-method-for-high-throughput-chemical-screening-caenorhabditis

Research

JoVE Journal

Biochemistry

8.7K Views.  The Buck Institute for Research on Aging. Hier beschrijven we een eenvoudig protocol voor het snel produceren van honderden nematode groei media agar, 96-Wells cultuur platen met een consistent aantal Caenorhhabditis elegans per putje. Deze culturen zijn nuttig voor de fenotypische screening van hele organismen. Hier richten we ons op met behulp van deze culturen op scherm chemicaliën voor Pro-levensduur effecten.

Video: A Simple Method for High Throughput Chemical Screening in 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

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.

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.

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

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

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.

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.

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 ...

Environment

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

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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 ...

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 ...

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.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

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