Name: biochemical and high throughput microscopic assessment of fat mass in caenorhabditis elegans
Uploaded: 2013-03-30T14:00:00
Duration: 16 min 7 s
Description: Watch this Scientific Journal Video about Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans at JoVE.com

We would like to thank Michael Kacergis for technical assistance and Lianfeng Wu for discussions and reading the manuscript. This work was supported by a career development award from the NIH/NIDDK (K08DK087941 to A.A.S.), a NIH/NIDDK training grant (5T32DK007028 to E.C.P.), the Ellison Medical Foundation New Scholar in Aging Award (to A.A.S), and the Charles H. Hood Foundation Child Health Research Award (to A.A.S.).

1. Preparation of Rectangular Amp/Tet (A/T) LB Agar Plates and NGM IPTG RNAi Plates
A/T plates should be prepared 1-4 weeks prior to use and stored in the dark at 4 &deg;C.
<ol>
	<li>For 1 L of media add 1 L deionized water, 32 g LB agar, 1.5 ml 2 M NaOH, and 2 g Bacto agar. Autoclave 40 min on liquid cycle. After cooling to 60 &deg;C, add 3 ml tetracycline and 0.5 ml ampicillin. Pour 45 ml of media into each plate.</li>
</ol>
Prepare 96-well RNAi plates 3 days up to 2 weeks in advance.
<ol start="2">
	<li>To pour 25 96-well RNAi plates, prepare 1 L of nematode growth media (NGM): 1 L deionized water, 3 g NaCl, 17 g agarose, and 2.5 g bactopeptone. Autoclave liquid cycle at 121 &deg;C for 40 min with a stir bar. Let cool, stirring on a hot plate set to 60 &deg;C.</li>
	<li>When cool to 60 &deg;C, add the following stock solutions: 1 ml 1 M MgSO4, 1 ml 5 mg/ml cholesterol, 25 ml 1 M KH2PO4 (pH 6.0), 1 ml 1 M CaCl2, 1 ml 200 mg/ml carbenicillin, and 5 ml 1 M IPTG (see Materials table for recipes).</li>
	<li>Dispense 150 &mu;l of RNAi media into each well of a flat-bottomed 96-well plate using an electronic multichannel pipettor. Avoid air bubbles. Keep media in a sterile stainless steel receptacle immersed in a 60 &deg;C water bath while dispensing.</li>
	<li>Keep RNAi plates level until agarose solidifies. Plates can be poured up to 2 weeks in advance and stored at 4 &deg;C.</li>
</ol>
Day 1
2. Stamp Frozen Library Plates onto Rectangular Amp/Tet (A/T) LB Agar Plates
<ol>
	<li>Warm A/T plates to room temperature, keeping them in the dark.</li>
	<li>Transfer 96-well RNAi library plates from -80 &deg;C to dry ice. Open the aluminum-sealed plate near a Bunsen burner.</li>
	<li>Sterilize a 96-pin replicator by immersing pins serially (10 sec each) in 10% bleach, deionized H2O, and 70% EtOH followed by passing pins through a flame. Repeat EtOH and flame step.</li>
	<li>Apply replicator to frozen 96-well RNAi glycerol stock, making sure each pin contacts the surface of each well.</li>
	<li>Stamp onto A/T plate, drawing a small circle with each pin without damaging agar surface.</li>
	<li>Re-seal and return library plates to -80 &deg;C. Incubate stamped A/T plates at 37 &deg;C overnight for bacterial growth.</li>
</ol>
Day 2
3. Grow RNAi Bacterial Clones Overnight in Deep-well Blocks
<ol>
	<li>Remove A/T plates from 37 &deg;C and confirm bacterial growth.</li>
	<li>Fill each well of a 96-well deep-well block with 1.5 ml of LB with 200 &mu;g/ml carbenicillin.</li>
	<li>Sterilize 96-pin replicator (see step 2.3).</li>
	<li>Apply replicator to the top of the A/T plate, making sure pins make contact with each bacterial RNAi clone. Lift replicator and immerse pins into the deep-well block. Swirl, and slowly remove, making certain not to contaminate adjacent wells.</li>
	<li>Seal block with breathe-easy film. Incubate overnight at 37 &deg;C with agitation (950 rpm in MT Infors Microtiteron II shaker, preferred, or 250 rpm in a 25 mm-throw shaking incubator).</li>
</ol>
Day 3
4. Seed RNAi Bacterial Clones onto 96-well NGM RNAi Plates
<ol>
	<li>Remove 96-well RNAi plates from 4 &deg;C and warm to room temperature.</li>
	<li>Remove deep-well blocks and centrifuge 10 min at 4,000 x g.</li>
	<li>Rapidly invert blocks into sink to drain media, and stamp inverted onto paper towel to eliminate excess media.</li>
	<li>Under a laminar flow hood, add 40 &mu;l of LB with 200 &mu;g/ml carbenicillin to each well and seal with sterile film.</li>
	<li>Return blocks to room temperature bacterial shaker for 10 min to resuspend pellets, or manually resuspend pellets by careful pipetting.</li>
	<li>In the laminar flow hood, remove sterile film and transfer bacteria from deep-well blocks onto 96-well RNAi plates. Add 40 &mu;l of resuspended bacteria to rows 'A' and 'H', and 35 &mu;l from all other rows. More liquid is added to edge wells of the 96-well RNAi plate to prevent overdrying and cracking of the agarose.</li>
	<li>Leave plates uncovered to dry in hood for 4-6 hr, inspecting regularly for dryness. Dry bacteria appear clear, not cloudy. Cover, invert plates and incubate at room temperature overnight.</li>
</ol>
5. Prepare a Synchronous Population of Worms
Two 10 cm plates filled with many gravid worms are used for egg preparation for every 6 96-well RNAi plates. Be sure worms are non-starved for at least two generations (7 days).
<ol>
	<li>Prepare a synchronous population of L1 as previously described20. For 20 ml of bleach solution, use 4 ml bleach, 1 ml NaOH (10 M), 5 ml deionized H2O, and 10 ml M9 buffer.</li>
	<li>Synchronize worms overnight in 10 ml M9 in a 15 ml sterile polypropylene tube with rotation at 20 &deg;C.</li>
</ol>
Day 4
6. Add Worms to RNAi Plates
<ol>
	<li>Centrifuge egg preps at 3,000 x g for 1 min. Aspirate supernatant, staying away from the pellet of L1 hatchlings. Resuspend in 5 ml M9 buffer with 0.0005% Triton X-100. Addition of the small amount of detergent prevents worms from sticking to the pipette and has no effect on fat staining.</li>
	<li>Count worms under a microscope. If necessary, adjust concentration to 10-15 worms per microliter.</li>
	<li>In the laminar flow hood, for each 96-well plate to be seeded, add 75 &mu;l of resuspended worms into each well of one row of a shallow-well assay block. Using a manual multichannel pipette, add 50-75 worms in 5 &mu;l into each well of the 96-well RNAi plates.</li>
	<li>Allow plates to dry in the hood for 30 to 60 min. When dry, under a microscope, worms should appear to be crawling, not thrashing.</li>
	<li>Grow worms on RNAi plates at 20 &deg;C for ~64 hr.</li>
</ol>
Day 7
7. Fix and Stain Worms in Nile Red
<ol>
	<li>Remove RNAi plates from incubator. Examine under microscope and record starved or thrashing (wet) wells to exclude from further analysis, as these conditions affect fat mass.</li>
	<li>Using an electronic repeat pipettor, add 150 &mu;l PBS with 0.01% triton X-100 to each well of an RNAi plate. With a manual multi-channel pipettor, mix and transfer liquid with worms to the corresponding row in a 96-deep-well PCR plate. Avoid disrupting the agar. Repeat for a total of 300 &mu;l worms in PBS per well.</li>
	<li>When four plates have been transferred, centrifuge plates at 500 rpm for 1 min.</li>
	<li>Aspirate supernatant using a 96-pin vacuum aspirator, leaving ~25 &mu;l liquid and worm pellet at the bottom of the well.</li>
	<li>Add 150 &mu;l of 40% isopropanol to each well to fix animals. Make certain that pipettor is on a high enough speed to mix worm pellet with 40% isopropanol. Incubate at room temperature for 3 min.</li>
	<li>Centrifuge plates uncovered at 500 rpm for 1 min.</li>
	<li>Aspirate supernatant using a 96-pin aspirator or manually, leaving only 25 &mu;l 40% isopropanol + worm pellet at the bottom of the well.</li>
	<li>To each well, add 150 &mu;l of Nile red staining solution prepared immediately before use: 6 &mu;l Nile red stock solution of 0.5 mg/ml in acetone per 1 ml of 40% isopropanol.</li>
	<li>Seal plate with non-sterile adhesive film and stain in the dark for at least 2 hr.</li>
</ol>
8. Wash and Image Worms on a High-throughput Microscope
<ol>
	<li>Centrifuge at 500 rpm for 1 min and aspirate all but 25 &mu;l of Nile red dye.</li>
	<li>Add 150 &mu;l of PBS with 0.01% triton X-100 to each well and store in the dark for at least 30 min.</li>
	<li>Centrifuge at 500 rpm for 1 min.</li>
	<li>Using a 12-channel pipettor, aspirate animals from the bottom of each well with 2 x 7 &mu;l quick strokes and dispense onto a 96-well Teflon-printed glass slide. Carefully, without removing worms, remove 9.5 &mu;l of PBS from each well of slide. If your glass slide does not fit directly in your microscope mount, a custom machined aluminum slide holder must be used to mount worms.</li>
	<li>Slowly lower cover slide onto 96-well slide. This step may take some practice to avoid bubbles or cross over of worms into other wells. Secure corners with adhesive tack.</li>
	<li>Image slide in bright field and fluorescence with a GFP/FITC filter set on an automated microscope. The lipid signal photobleaches easily so it should be imaged in a systematic way in all wells, not manually, as photobleaching will lead to artificial differences between wells. For more details on analysis, please see Representative Results and Discussion.</li>
</ol>
9. Biochemical Lipid Determination Using Solid Phase Extraction (SPE) and Gas Chromatography-Mass Spectrometry (GC/MS)
For validation of lipid levels based on Nile red fluorescence, gas chromatography followed by mass spectrometry analysis (GC/MS) can be performed on fatty acid methyl esters (FAME) derived from triacylglycerols (TAG) and phospholipids (PL) from worm pellets. Steps involving organics should be performed under a fume hood. Care should be taken that lipids are kept under nitrogen protected from light during extended storage or incubation steps as they are very vulnerable to oxidation. Worm pellets from 2,500-5,000 synchronous animals are used as input, prepared exactly as above except that animals are grown on 10 cm RNAi plates.
<ol>
	<li>Wash 2,500-5,000 worms from each 10 cm plate with 10 ml M9 buffer into a 15 ml conical polypropylene tube. Pellet worms at 500 x g for one minute, and wash pellet again with 10 ml M9 buffer. Pellet animals at 500 x g. Aspirate leaving 250 &mu;l total volume, and either freeze on liquid nitrogen and store at -80 &deg;C under nitrogen for later processing or proceed directly.</li>
	<li>The worm pellet may be taken directly to step 9.3 if triglyceride mass is to be normalized to phospholipid mass8,13,21 . However, if the investigator desires normalization to protein content or DNA content, the worm pellet should be sonicated on the highest intensity in a bath sonicator for 10 min, 30 sec on, 30 sec off, at 4 &deg;C. If protein or DNA normalization is desired, remove a 5 &mu;l aliquot and determine total protein concentration using a Bradford reagent or similar or total DNA content after column purification using a UV spectrophotometer.</li>
	<li>Add worms to 1.5 ml 2:1 chloroform:methanol in a threaded borosilicate glass tube. All transfer of organic solvents should be done using glass pipettes.</li>
	<li>Using a wiretrol 50 &mu;l pipette, add 25 nM phospholipid standard in 50 &mu;l, and 16.7 nM triglyceride standard in 50 &mu;l. Both standards should be dissolved in 2:1 chloroform:methanol, capped tightly, and stored under nitrogen protected from light in a -80 &deg;C freezer to avoid oxidation.</li>
	<li>Cap with phenolic cap and vortex. Extract for 1 hr at RT, vortexing every 15 min.</li>
	<li>Centrifuge at 1,000 rpm for 5 min. Transfer lower organic phase without carcasses to a borosilicate culture tube.</li>
	<li>Add 0.3 ml 0.9% NaCl, vortex and centrifuge at 1,000 rpm for 5 min.</li>
	<li>Transfer bottom organic layer to a fresh borosilicate culture tube, making sure not to transfer over any of the aqueous phase.</li>
	<li>Dry organic phase under nitrogen. To begin solid phase extraction (SPE) resuspend dried lipid in 1 ml chloroform. Alternatively, for detailed separation and analysis of distinct lipid classes, including phospholipid classes, glycolipids, sphingolipids, diacylglycerols, triacylglycerols, free fatty acids, and cholesterol esters, thin layer chromatography as pioneered by the Watts lab in C. elegans22 may be used in place of SPE. Lipids may also be separated by HPLC and fractions analyzed by GCMS . Sophisticated methods of whole-lipidome profiling by liquid-chromatography/MS (LCMS) can also be used23-26.</li>
	<li>Assemble silica SPE column on SPE manifold. Pre-equilibrate column with 3 ml chloroform. Allow solvent to flow by gravity.</li>
	<li>Load lipid sample onto SPE column, collecting flow-through in a threaded borosilicate tube as part of the first fraction (TAG fraction). Most neutral lipids will come through in this first 1 ml flow-through. Add 3 ml chloroform to fully elute TAG fraction, collecting flow-through in the TAG fraction tube.</li>
	<li>Remove and cap the first collection tube, and replace with a clean collection tube. Elute glycosphingolipids with 5 ml of 9:1 acetone:methanol. We do not routinely analyze the fatty acid composition of this fraction, however, it may contain valuable information and can be saved for preparation of methyl esters and free sphingoid bases for further analysis utilizing specialized procedures distinct from those for TAG and PL as previously described 27.</li>
	<li>Replace glycosphingolipid collection tube with a second threaded borosilicate collection tube. Elute phospholipids with 3 ml methanol (PL fraction).</li>
	<li>Fractions can be stored at -80 &deg;C at this point capped tightly under nitrogen. Dry purified lipids under nitrogen. To speed drying, in a dry heating block at 33 &deg;C. Never store lipids in dried form, as they are especially susceptible to oxidation in the dried state.</li>
	<li>Resuspend dried lipid fractions in 2 ml 2% H2SO4 in methanol by vortexing and incubate capped tightly overnight in a 55 &deg;C water bath to create FAMEs. Care must be taken to get absolutely no water in the FAME reaction as this will greatly inhibit derivatization of lipids. We have found more efficient derivatization by overnight incubation and the literature suggests less lipid oxidation occurs at lower temperatures during FAME preparation28. An alternate and more rapid method is to use 2 ml 2.5% H2SO4 in methanol and derivatize for one hour at 70 or 80 &deg;C21,22,29 .</li>
	<li>The next morning, remove from bath, and allow to cool. Add 1.5 ml H2O (to quench the reaction) and then 0.3 ml hexane (to extract the FAMEs) to each TAG and PL fraction. Shake vigorously and centrifuge at 1,000 rpm for 5 min.</li>
	<li>Very carefully remove only top hexane layer using a standard pipette or Pasteur pipette. Dispense into autosampler tube. Make certain not to include any acidified methanol as this will destroy the GC column and MSD.</li>
	<li>Cap and load samples onto GC/MS. The sample is transferred in hexane to a micro-insert Agilent vial capped with a PTFE-silicone-PTFE screw cap. It is transferred to the Agilent GCMS model 6890/5973N outfitted with a Supelcowax-10 (24079) 30 meter, 250 micron fused silica capillary column. One microliter is injected into a splitless inlet set at 250 &deg;C and 13.33 P.S.I. Ultrapure Helium is used a carrier gas. The protocol run is as follows at a constant 1 ml per min:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Instruction</td>
			<td>Goal Temperature</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Hold 2 min</td>
			<td>150 &deg;C</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Ramp 10 &deg;C per min</td>
			<td>200 &deg;C</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Hold 4 min</td>
			<td>200 &deg;C</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Ramp 5 &deg;C per min</td>
			<td>240 &deg;C</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Hold 3 min</td>
			<td>240 &deg;C</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Ramp 10 &deg;C per min</td>
			<td>270 &deg;C</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Hold 5 min</td>
			<td>270 &deg;C</td>
		</tr>
	</tbody>
</table>
A 3 min solvent delay is used at the MSD to minimize wear on the filament. Thereafter, masses between 50 and 550 Daltons are detected with the MS quadruple set at 150 &deg;C and the ms source set at 230 &deg;C, with a thermal auxiliary temperature of 270 &deg;C.

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P., 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=Lipid+profiling+identifies+a+triacylglycerol+signature+of+insulin+resistance+and+improves+diabetes+prediction+in+humans.">Lipid profiling identifies a triacylglycerol signature of insulin resistance and improves diabetes prediction in humans.</a> The Journal of Clinical Investigation. 121, 1402-1411 (2011).</li><li>Lewis, G. D., 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=Metabolic+signatures+of+exercise+in+human+plasma.">Metabolic signatures of exercise in human plasma.</a> Science translational medicine. 2, 33-37 (2010).</li><li>Rhee, E. P., 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=Metabolite+profiling+identifies+markers+of+uremia.">Metabolite profiling identifies markers of uremia.</a> Journal of the American Society of Nephrology : JASN. 21, 1041-1051 (2010).</li><li>Gerdt, S., Lochnit, G., Dennis, R. D., Geyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structural+analysis+of+three+neutral+glycosphingolipids+from+a+mixed+population+of+Caenorhabditis+elegans+(Nematoda:Rhabditida).">Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda:Rhabditida).</a> Glycobiology. 7, 265-275 (1997).</li><li>Christie, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Lipid Methodology - Two. , 69-111 (1993).</li><li>Watts, J. L., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+polyunsaturated+fatty+acid+synthesis+in+Caenorhabditis+elegans.">Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5854-5859 (2002).</li><li>Wahlby, C., 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=An+image+analysis+toolbox+for+high-throughput+C.+elegans+assays.">An image analysis toolbox for high-throughput C. elegans assays.</a> Nat. Methods. , (2012).</li><li>Fowler, S. D., Brown, W. J., Warfel, J., Greenspan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+nile+red+for+the+rapid+in+situ+quantitation+of+lipids+on+thin-layer+chromatograms.">Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms.</a> J. Lipid Res. 28, 1225-1232 (1987).</li><li>Squiban, B., Belougne, J., Ewbank, J., Zugasti, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+Automated+High-throughput+Genome-wide+RNAi+Screens+in+C.+elegans.">Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans.</a> J. Vis. Exp. (60), e3448 (2012).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+Screening+and+Biosensing+with+Fluorescent+C.+elegans+Strains.">High-throughput Screening and Biosensing with Fluorescent C. elegans Strains.</a> J. Vis. Exp. (51), e2745 (2011).</li><li>Comizio, R., 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=Total+body+lipid+and+triglyceride+response+to+energy+deficit:+relevance+to+body+composition+models.">Total body lipid and triglyceride response to energy deficit: relevance to body composition models.</a> The American journal of physiology. 274, E860-E866 (1998).</li><li>Lee, R. F., Paffenhofer, G. A., Nevenzel, J. C., Benson, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabolism+of+wax+ters+and+other+lipids+by+the+marine+copepod,+Calanus+helgolandicus.">The metabolism of wax ters and other lipids by the marine copepod, Calanus helgolandicus.</a> J. Lipid Res. 11, 237-240 (1970).</li><li>Lee, S. S., 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=Requirement+of+PPARalpha+in+maintaining+phospholipid+and+triacylglycerol+homeostasis+during+energy+deprivation.">Requirement of PPARalpha in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation.</a> J. Lipid Res. 45, 2025-2037 (2004).</li><li>Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluid+lipid-globular+protein+mosaic+model+of+membrane+structure.">A fluid lipid-globular protein mosaic model of membrane structure.</a> Annals of the New York Academy of Sciences. 195, 16-23 (1972).</li><li>Singer, S. J., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluid+mosaic+model+of+the+structure+of+cell+membranes.">The fluid mosaic model of the structure of cell membranes.</a> Science. 175, 720-731 (1972).</li><li>Turkish, A. R., Sturley, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+neutral+lipid+biosynthesis:+an+evolutionary+perspective.">The genetics of neutral lipid biosynthesis: an evolutionary perspective.</a> American Journal of Physiology. Endocrinology and Metabolism. 297, E19-E27 (2009).</li></ol>

The authors have no conflicting interests to disclose.

The presented protocol allows for rapid, large scale screening of lipid levels in C. elegans, which can be easily adapted to high throughput screens. Unlike previously used methods, which involve a lengthy fixation step in paraformaldehyde followed by several freeze-thaw cycles8,10 , our protocol requires a simple 3-min fixation in isopropanol. We have found that by staining C. elegans in 40% isopropanol, they become freely permeable to Nile red, without the use of additional freeze-thaw or fixation steps. Also, as Nile red selectively fluoresces in hydrophobic compartments in the green spectrum18,19,31 , no destaining is necessary. In total, animals can be taken from RNAi plates to stained and ready to image animals in just over 2.5 hr. This method can be performed relatively inexpensively and with high reproducibility. The method does not depend upon the ability of C. elegans to uptake or concentrate the dye, and therefore does not have the same limitations as feeding based methods using vital dyes. Given reproducibility and precision of measurements, the method is suitable for screening large numbers of gene inactivations by RNAi. If quantitation of lipid levels is desired based upon fixative Nile red staining, we recommend the use of 4-6 biological replicates with the appropriate controls and statistical testing applied.
It is likely that many gene inactivations leading to small animals (due to RNAi clones causing delayed or arrested development) would come out of a screen for fat regulators, regardless of any connection to fat metabolism. While the image analysis program we utilized accounts for worms of different sizes by normalizing the fluorescence intensity to the worm area, individual researchers may also want to consider comparing the fat staining levels of small animals to wild type animals of a similar developmental stage. In the case of small or developmentally arrested animals with apparent low fat mass, we suggest that no single modality is sufficient to demonstrate altered lipid metabolism. Several follow-up experiments, including SPE-GCMS, could be required to ascertain the lipid-regulatory function of these genes. For known metabolic regulator fasn-1, SPE-GCMS analysis compared to developmentally matched N2 L2 control animals was necessary to confirm the apparent low fat phenotype found when compared to adult control animals. While Nile red fat staining indicated lower fat levels relative to control adult RNAi, when compared to stage-matched L2 control RNAi animals, no apparent difference was seen. Accordingly, use of a second method, i.e. biochemical quantification of the TAG/PL ratio was necessary to determine that fasn-1 is a true regulator of fat mass, as fasn-1 RNAi is biochemically distinguishable from stage-matched L2 control RNAi animals.
While we present imaging and processing for a high-throughput platform, a conventional upright microscope can easily be used to capture images from worms mounted on agarose pads20 or on conventional sized Teflon-printed 8 to 12 well microscope slides. The only limitation is that as the green fluorescence of lipid droplets stained with Nile red photobleaches quickly, systematic, uniform exposure conditions need to be used to ensure comparability between images. We find that a fully automated microscope with uniform image acquisition conditions works best to this effect.
One of the great strengths of this protocol is that after images are collected they may be saved for future re-analysis. The same images could be used to determine body size in addition to fat content. Moreover, as computational programs become more advanced, there is the prospect for single worm analysis, generating statistically significant results from a single well. Thus our method that utilizes high-throughput microscopy has some advantages over published screen methodologies that use a Copas Biosort to quantify fluorescent signals32,33 . However, the methodologies are definitely complimentary.
Precise, biochemical determination of lipid content by SPE and GC/MS confirms the Nile red staining of the major fat stores in C. elegans. Although the absolute quantitation achieved from fixative Nile red staining and biochemical lipid determination often do not match perfectly, both methods produce highly reproducible, statistically significant results that agree qualitatively. In addition, this method can be tailored to meet the specific needs of individual researchers who may require further analysis of the separate classes of glycosphingolipids, phospholipids, or triglycerides present in various samples. It should be noted that other groups have used technology other than SPE to separate lipid classes prior to GC/MS22. Thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) has advantages over SPE in that they might be able to better separate distinct neutral lipids (e.g. TAG, diacylglycerols, free fatty acids, and cholesterol esters) from polar lipids (phosphatidylcholine, phosphatidylethanolamine) and glycol-sphingolipids. We choose SPE because it requires a minimum of starting material (2,500-5,000 worms), it emphasizes the major long-term energy stores, TAGs, which make up most of the neutral lipid fraction4, and is rapid, requiring no specialized equipment or plates. It should be emphasized that based upon standard mixtures, SPE completely separates TAGs from PLs, and is highly reproducible and reliable (see Figure 5A). While quantitation and analysis of fatty acid methyl esters requires a GC/MS, this equipment is commonly available, and if not locally, samples may be generated as above and stored at -80 &deg;C under nitrogen until analysis by a collaborator or commercial GC/MS service is arranged.
The GCMS output contains high-resolution data on each fatty acid within each lipid species. This permits the determination of detailed differences between samples compared. As an example, the investigator could determine whether the levels of certain fatty acids were altered in abundance more significantly than others in daf-2, lpd-3 or fasn-1 RNAi versus vector control. This enormously powerful tool can therefore provide detailed information on which lipid species are altered in abundance with any experimental manipulation.
For our analysis, we most often normalize TAG mass to PL mass. While protein or DNA could also be used to normalize lipid mass following determination from an aliquot of sonicated worm pellet, we find that these methods are subject to introduced human error during all pipetting steps and in sample recovery from each extraction. It is for this reason that we chose to instead use PL mass as our normalization factor. Unnatural standards introduced at the start of the protocol (tritridecanoin for TAG, 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) for PL) are carried through all steps of the solid phase extraction and FAME preparation, and guarantee quantitative and representative recovery of both TAG and PL fractions. The same guarantee cannot be made if investigators use protein or DNA to normalize TAG mass. We believe PL to be the most robust gauge by which to compare TAG levels across samples, as it has been previously shown that PL is only minutely altered by the same stimulus effecting large mass shifts in TAG levels, e.g. extended starvation or increased exercise34-36. PL are thought to play a structural role, ensuring membrane fluidity and cellular integrity and signaling roles rather than as energy storage37-39. In our hands, the TAG/PL ratio most closely matches what is obtained by fixative-based lipid staining with Nile red, and agrees with that found by other groups21. An alternative strategy is used by the Watts laboratory, where the percentage of lipid TAG is calculated versus total lipids9,22,29 .
The use of non-native types of TAG and PL standard ensures that no native fatty acids will be included in the normalization calculation. The choice of chain length is purely based upon the need of these standards to not interfere with the mass spectra of native fatty acids. We have found that fatty acids of 13:0 and 17:0 serve best to this purpose. It is possible, given the different chemical properties of shorter chain fatty acids, that our unnatural fatty acid standards might not be representative of recovery of all chain-length fatty acids or chains with different saturation indices. Thus it is possible that during SPE or GCMS we might see differences in the efficiency of purification or detection between lipids of different chain lengths. Based upon this and the different ionization of different fatty acids in the GCMS, it is not possible to make an absolute statement about the abundance of any given fatty acid. Therefore, we only compare between samples within a single experiment abundances of any given fatty acid. If one wanted to absolutely quantitate a fatty acid, one manner this could be achieved would be to run a sample prepared as above spiked with a known amount of a stably isotopically labeled 13C version of that or similar fatty acid, so that it could be distinguished based upon mass of the parent ion in the MSD, for example. Given that we are only comparing relative amounts of lipid classes or any given fatty acid, our 13:0 TAG and 17:0 PL standards serve this purpose adequately, at a minimum of cost, to assure adequate and representative recovery of each lipid class.
Until this point, there has been a lack of consensus as to the interpretation of certain results obtained by various methodologies, and to what the prevailing methodologies should be. Overall it should be noted that no one method in isolation is ideally suited to study fat metabolism in C. elegans. However, by using multiple screening and validation modalities, one can achieve confidence in data obtained on lipid regulatory genes. Thus, we intend for our protocol to serve as a benchmark for future work in the field of C. elegans fat research.

Conservation of metabolic pathways between humans and the nematode Caenorhabditis elegans makes it a powerful model organism for the study of obesity. While C. elegans do not have adipocytes dedicated to fat storage like in mammals, they do store triglycerides in lipid droplets1 and possess many of the same regulators of fat storage and energy use2,3. To assay for fat levels in C. elegans, several methods have been proposed with varying levels of ease and success. The once common use of fluorescent, vital dyes4-7 was recently called into question, and these reagents proved to be staining a compartment distinct from the main fat storage depot of C. elegans1,8-11 . Fixative-based non-fluorescent dyes, such as Sudan black and Oil-red-O, while successful at highlighting lipid droplets in the worm12-14, do not have as large a dynamic range for quantitation as fluorescent dyes8,13,15. Furthermore, current methods of fixation for both fluorescent and non-fluorescent dyes are time-consuming and involve multiple freeze-thaw steps and/or the use of toxic chemicals1,9,10 . The use of specific BODIPY-labeled lipid analogs has been reported to highlight lipid droplets when fed to living worms with short-term labeling15. However this relies on uptake of the dye and is therefore biased by C. elegans handling and metabolism of BODIPY fatty acid analogs. An established alternative to lipid-staining dyes is label-free, coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS) microscopy, which take advantage of the characteristic vibrational properties of lipid molecules to visualize fat stores10,16,17 . However, expensive specialized equipment is necessary for this method, and throughput is low at best.
To fulfill a need for rapid, scalable analysis of neutral lipid stores in C. elegans metabolic research, we introduce a novel, highly reproducible method of fixative-based Nile red lipid staining. Spectral and physicochemical properties of the lipophilic dye Nile red induce a yellow-gold-spectral shift in its excitation-emission peak, allowing it to fluoresce in the green emission spectrum only when in a lipid-rich environment, but not in more polar environments18,19 . Thus lipid droplets can be detected after simple staining by the use of a green fluorescent protein (GFP) filter set for fluorescence microscopy. The ease and affordability of this technique make it ideally suited for high throughput screens. We also demonstrate a paired, rigorous method for biochemical determination of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry. Biochemical lipid measurement correlates with Nile red-based microscopic determination of C. elegans fat mass, and should be used as a confirmation of findings made with microscopic lipid determination.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>96-pin replicator</td>
 <td>Nunc</td>
 <td>250520</td>
 <td></td>
 </tr>
 <tr>
 <td>LB Agar</td>
 <td>US Biologicals</td>
 <td>L1500</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>16500500</td>
 <td></td>
 </tr>
 <tr>
 <td>Mechanical Pipettors</td>
 <td>Biohit M Line</td>
 <td>M10, M100, M300</td>
 <td></td>
 </tr>
 <tr>
 <td>Electronic Pipettor</td>
 <td>Biohit E Line</td>
 <td>E1200</td>
 <td></td>
 </tr>
 <tr>
 <td>1 M KH2PO4</td>
 <td>Sigma</td>
 <td>P0662</td>
 <td>pH 6.0 Sterilized by Autoclaving</td>
 </tr>
 <tr>
 <td>1 M MgSO4</td>
 <td>Sigma</td>
 <td>M2643</td>
 <td>Sterilized by Autoclaving</td>
 </tr>
 <tr>
 <td>1 M CaCl2</td>
 <td>Sigma</td>
 <td>C2661</td>
 <td>Sterilized by Autoclaving</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma</td>
 <td>S5886</td>
 <td></td>
 </tr>
 <tr>
 <td>5 mg/ml Cholesterol</td>
 <td>Sigma</td>
 <td>C8667</td>
 <td>In 100% Ethanol</td>
 </tr>
 <tr>
 <td>200 mg/ml Carbenicillin</td>
 <td>US Biologicals</td>
 <td>C1100</td>
 <td>Filter Sterilized 0.2 &mu; </td>
 </tr>
 <tr>
 <td>1 M IPTG</td>
 <td>US Biologicals</td>
 <td>I8500</td>
 <td>Filter Sterilized 0.2 &mu; </td>
 </tr>
 <tr>
 <td>100 mg/ml Ampicillin</td>
 <td></td>
 <td></td>
 <td>Filter Sterilized 0.2 &mu; </td>
 </tr>
 <tr>
 <td>5 mg/ml Tetracycline</td>
 <td></td>
 <td></td>
 <td>In 100% Ethanol</td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>BD</td>
 <td>214040</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well TC Plates</td>
 <td>BD-Falcon</td>
 <td>353072</td>
 <td>For 96-well RNAi</td>
 </tr>
 <tr>
 <td>Rectangular A/T Plates</td>
 <td>Thermo Scientific</td>
 <td>242811</td>
 <td>For stamping RNAi</td>
 </tr>
 <tr>
 <td>LB Media</td>
 <td>US Biologicals</td>
 <td>L1530</td>
 <td>Prepared as per manufacturer instructions</td>
 </tr>
 <tr>
 <td>96-well Deep Well Assay Blocks</td>
 <td>Corning</td>
 <td>Costar 3960</td>
 <td>Rectangular V-bottom assay block</td>
 </tr>
 <tr>
 <td>96-well Shallow Well Assay Block</td>
 <td>Corning</td>
 <td>Costar 3956</td>
 <td>Round V-bottom assay block</td>
 </tr>
 <tr>
 <td>Sterile Sealing Film</td>
 <td>VWR</td>
 <td>89134-432</td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum Sealing Film</td>
 <td>Corning</td>
 <td>Costar 6570</td>
 <td>Thermowell Sealing tape for 96-well plates</td>
 </tr>
 <tr>
 <td>M9 Buffer</td>
 <td>See Hope et al.20 </td>
 <td></td>
 <td>See recipe below</td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Bio-Rad</td>
 <td>161-0407</td>
 <td></td>
 </tr>
 <tr>
 <td>Nile red</td>
 <td>Invitrogen</td>
 <td>N-1142</td>
 <td></td>
 </tr>
 <tr>
 <td>Isopropanol</td>
 <td>Fisher</td>
 <td>BP2632-4</td>
 <td></td>
 </tr>
 <tr>
 <td>96-pin aspirator</td>
 <td>VP Scientific</td>
 <td>VP177A-1</td>
 <td></td>
 </tr>
 <tr>
 <td>96-well Teflon Masked Microscope Slides</td>
 <td>Thermo Scientific</td>
 <td>Custom</td>
 <td>Can also order Trevigen 96-well Cometslide</td>
 </tr>
 <tr>
 <td>96-well Gold-Seal Coverslides</td>
 <td>Thermo Scientific</td>
 <td>Custom</td>
 <td>Can also use second 96-well Cometslide </td>
 </tr>
 <tr>
 <td>Deep-well PCR plate</td>
 <td>VWR</td>
 <td>89049-178</td>
 <td></td>
 </tr>
 <tr>
 <td>Non-sterile adhesive film</td>
 <td>VWR</td>
 <td>60941-070</td>
 <td></td>
 </tr>
 <tr>
 <td>High-Throughput Microscope</td>
 <td>Leica</td>
 <td>DM6000 fully automated with Prior 96-well stage</td>
 <td>With Chroma 49002 GFP filter set and 2x objective</td>
 </tr>
 <tr>
 <td>Bath Sonicator</td>
 <td>Diagenode</td>
 <td>Bioruptor XL</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma</td>
 <td>366927-4L</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher</td>
 <td>Optima F456-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Phospholipid Standard</td>
 <td>Avanti Polar Lipids</td>
 <td>830456P</td>
 <td>1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) </td>
 </tr>
 <tr>
 <td>Triglyceride Standard</td>
 <td>NuChek Prep</td>
 <td>T-135</td>
 <td>tritridecanoin</td>
 </tr>
 <tr>
 <td>Wiretrol 50 &mu;l Pipet</td>
 <td>Fisher</td>
 <td>21-176-3A</td>
 <td>Drummond Scientfic</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Sigma</td>
 <td>320110-4L</td>
 <td></td>
 </tr>
 <tr>
 <td>Sulfuric Acid</td>
 <td>Fisher</td>
 <td>A300-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Hexane</td>
 <td>Fisher</td>
 <td>Optima H306-1</td>
 <td></td>
 </tr>
 <tr>
 <td>SPE Silica Column</td>
 <td>Thermo Scientific</td>
 <td>60108-317</td>
 <td>Hypersep SI, 100 mg resin bed </td>
 </tr>
 <tr>
 <td>Solid Phase Chromatography Manifold</td>
 <td>Sigma </td>
 <td>57265</td>
 <td>Supelco Visiprep 24-DL </td>
 </tr>
 <tr>
 <td>Borosilicate Pipets</td>
 <td>Fisher</td>
 <td>13-678-27F</td>
 <td>10 ml size</td>
 </tr>
 <tr>
 <td>Borosilicate Pasteur Pipets</td>
 <td>Fisher</td>
 <td>13-678-8B</td>
 <td></td>
 </tr>
 <tr>
 <td>Borosilicate Culture Tubes</td>
 <td>Fisher</td>
 <td>14-961-27</td>
 <td></td>
 </tr>
 <tr>
 <td>Threaded Borosilicate Tubes</td>
 <td>VWR</td>
 <td>14235-460 </td>
 <td>8 ml capacity</td>
 </tr>
 <tr>
 <td>Phenolic Caps for Culture Tubes</td>
 <td>Fisher</td>
 <td>14-823-211</td>
 <td></td>
 </tr>
 <tr>
 <td>GC/MS Autosampler Vials</td>
 <td>Agilent</td>
 <td>5188-6592</td>
 <td></td>
 </tr>
 <tr>
 <td>Caps for Autosampler Vials</td>
 <td>Agilent</td>
 <td>5185-5861</td>
 <td></td>
 </tr>
 <tr>
 <td>GC/MS</td>
 <td>Agilent</td>
 <td>6890 GC / 5973 MSD</td>
 <td>Outfitted with a Supelcowax-10 column</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Nematode Growth Media (NGM): for 25 96-well RNAi plates, prepare 1 L of NGM:
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 </tr>
 <tr>
 <td>Deionized water</td>
 <td>1 L</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>3 g</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>17 g</td>
 </tr>
 <tr>
 <td>Bacto peptone</td>
 <td>2.5 g</td>
 </tr>
 <tr>
 <td colspan="2">Autoclave liquid cycle 121 &deg;C for 40 min with a stir bar. Allow to cool to 60 &deg;C stirring on a hot plate set at 60 &deg;C. Then add the following:</td>
 </tr>
 <tr>
 <td>1 M MgSO4</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>5 mg/ml cholesterol</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>1 M KH2PO4 (pH 6.0)</td>
 <td>25 ml</td>
 </tr>
 <tr>
 <td>1 M CaCl2</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>200 mg/ml carbenicillin</td>
 <td>1 ml</td>
 </tr>
 <tr>
 <td>1 M IPTG</td>
 <td>5 ml</td>
 </tr>
 <tr>
 <td colspan="2">Dispense into 96-well RNAi plates. Store at 4 &deg;C until use, up to 2 weeks.</td>
 </tr>
 </tbody>
</table>
Rectangular Ampicillin/Tetracycline LB Agar Plates (A/T): for 20 A/T plates, prepare 1 L of media:
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 </tr>
 <tr>
 <td>Deionized water</td>
 <td>1 L</td>
 </tr>
 <tr>
 <td>LB agar (US Biologicals)</td>
 <td>32 g</td>
 </tr>
 <tr>
 <td>2 M NaOH</td>
 <td>1.5 ml</td>
 </tr>
 <tr>
 <td>Bacto agar</td>
 <td>2.5 g</td>
 </tr>
 <tr>
 <td colspan="2">Autoclave liquid cycle 121 &deg;C for 40 min with a stir bar. Allow to cool to 60 &deg;C stirring on a hot plate set at 60 &deg;C. Then add the following:</td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>3 ml</td>
 </tr>
 <tr>
 <td>100 mg/ml Ampicillin</td>
 <td>0.5 ml</td>
 </tr>
 <tr>
 <td colspan="2">Dispense 45 ml into each plate. Store in dark at 4 &deg;C until use, up to 4 weeks.</td>
 </tr>
 </tbody>
</table>
LB bacterial growth media: for 4 L, sufficient for growing and resuspending 25 96-well blocks
Mix 62 g LB powder (US Biologicals), 4 L distilled water, and 4.8 ml 2 M NaOH. Autoclave in 1 L or 0.5 L aliquots, liquid cycle, 40 min. Can be stored up to 1 year in airtight bottles at room temperature. Discard if cloudy.
100 mg/ml Ampicillin
Mix 5 g ampicillin powder and deionized water to a total volume of 50 ml. Filter sterilize 0.2 &mu;, dispense in 1 ml aliquots and freeze at -20 &deg;C until use.
5 mg/ml Tetracycline
Add 1.25 g tetracycline powder to 250 ml 100% ethanol. Mix well until fully dissolved and store at -20 &deg;C in a light-tight container.
200 mg/ml Carbenicillin
Mix 10 g carbenicillin powder and deionized water to a total volume of 50 ml. Filter sterilize 0.2 &mu;, dispense in 1 ml aliquots and freeze at -20 &deg;C until use.
5 mg/ml Cholesterol
Add 1.25 g cholesterol to 250 ml 100% ethanol. Mix well and store at 4 &deg;C for up to 6 months. 
1 M MgSO4
Dissolve 120.37 g MgSO4 in 800 ml deionized water. Bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
1 M CaCl2
Dissolve 110.98 g CaCl2 in 800 ml deionized water. Bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
1 M KH2PO4, pH 6.0
Dissolve 136.09 g KH2PO4 in 800 ml deionized water. pH to 6.0 and bring final volume to 1 L. Autoclave 40 min liquid cycle and store at room temperature.
M9 Buffer: for 1 L&#x2014;we routinely make 8-10 L batches
Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4 in 900 ml. Bring final volume to 1 L. Aliquot in 100-500 ml bottles and autoclave 40 min on liquid cycle and store at room temperature.
10 N NaOH
Dissolve 400 g NaOH in 800 ml deionized water, allow to cool and bring to a final volume of 1 L. Store in 50 ml tubes at room temperature.
1 M IPTG
Add 23.81 g IPTG to deionized water and bring total volume to 100 ml. Filter sterilize 0.2 &mu;, dispense in 1 ml aliquots and freeze at -20 &deg;C until use.
Egg Preparation Bleach/NaOH Solution: 20 ml sufficient for one egg preparation
Add 4 ml bleach, 1 ml 10 N NaOH, 5 ml H2O, and 10 ml M9 buffer to a 50 ml polypropylene conical tube. Make immediately before use.
Nile red stock solution
Add 50 ml 100% Acetone to 25 mg Nile red powder. Mix well and store at room temperature in light-tight parafilmed container. Can be stored for months in this manner.
Nile red staining solution: for 500 ml, sufficient for staining 30 x 96-well plates
Add 3 ml Nile red stock solution to 500 ml 40% v/v isopropanol. Store at room temperature in foil-wrapped glass bottle. Prepare immediately before use.
Phospholipid Standard
Weigh 7.6 mg phospholipid standard, 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol), in a glass beaker. Dissolve in 20 ml 2:1 chloroform:methanol (25 nanomoles per 50 &mu;l). Keep covered with aluminum foil until use. Standard can also be kept at -80 &deg;C in an airtight glass vial with phenolic cap for 1 year.
Triglyceride Standard
Weigh 6.8 mg triglyceride standard, tritridecanoin, into a glass beaker. Dissolve in 30 ml 2:1 chloroform:methanol (16.7 nanomoles per 50 &mu;l). Keep covered with aluminum foil until use. Standard can also be kept at -80 &deg;C in an airtight glass vial with phenolic cap for 1 year.</td>
 </tr>
 </tbody>
</table>

biochemical and high throughput microscopic assessment of fat mass in caenorhabditis elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

An example of an imaged plate that has been taken through the fat staining workflow is shown in Figure 1. Specific controls for high fat (daf-2 insulin receptor RNAi), low fat (lpd-3 protein required for lipid storage granules in the gut RNAi), small and low fat (fasn-1 fatty acid synthase RNAi), and empty vector (control) show the variations in fluorescence intensity according to the fat levels of the animals, and the corresponding lipid amounts determined by GC/MS analysis (Figure 2). Note that gene inactivations leading to developmentally arrested, small animals will often appear to have much lower fat than adult control animals, irrespective of any connection to fat metabolism (Figure 3). Secondary analyses, including staining analysis and lipid biochemistry with SPE-GCMS of wild-type control animals of a similar developmental stage must be done to ensure the validity of small or developmentally arrested positive hits. In the case of fasn-1 RNAi (Figure 2), animals arrest in either the L2 or L3 larval stage, and have fat staining lower than adult control RNAi, but similar in abundance to L2-L3 stage control RNAi animals (fat stain percent of adult control RNAi: 49.9 &plusmn; 4.5% for fasn-1 RNAi and 20.2 &plusmn; 2.1% for L2 control RNAi, and 64.7 &plusmn; 1.3 for L3 control RNAi). Alternatively, biochemical analysis indicated a lower TAG/PL ratio than L2 stage matched N2 control animals (TAG/PL: 22.5 &plusmn; 2.9 for fasn-1 RNAi and 46.1 &plusmn; 3.4 for stage-matched control RNAi, P &le; 0.05). Thus in this instance it can be confirmed by biochemical analysis that fasn-1 has a role in lipid storage rather than just a stage-specific reduction in fat mass.
The follow-up analysis of imaged worms relies heavily on a computational platform such as Cell Profiler using the WormToolbox30. For smaller numbers of images, a publicly available image analysis platform such as ImageJ, freely available from the NIH, may be used. For our analysis, unique Matlab scripts were used first to identify the well, and subsequently to exclude empty wells or those with bubbles, and distinguish small debris from worms. Manual quality control was necessary for images flagged for exclusion for the reasons mentioned above. We found empirically that 90th percentile intensity of worm pixels across a well, normalized to worm area across a well, provided consistent metric of staining intensity at all stages of worm development (Figure 3). Integrating total fat mass over the area of the worm, on the contrary, would tend to score small bright worms equivalently with large dim worms. Because our method does not integrate total fluorescent signal over the area of the worm, in taking an intensity percentile, changes in worm size per se do not bias the intensity measured. Instead, differences in the pixel intensity distribution are measured. However, in spite of this, pronounced differences are seen in staining pixel intensity distribution between animals of different developmental stages, so it is important to study the effect of RNAi to any gene against animals of a comparable developmental stage (Figure 3). Average variability between replicates is shown in Figure 4 indicating high reproducibility of the fat staining method. Note that the strength of the method is highly dependent on obtaining quality images for each plate, under uniform illumination conditions, using identical exposure times, and with minimal bubbles, debris, or crossover of worms into other wells.
SPE was performed on pre-mixed, purified standards to determine the degree to which TAGs could be separated from PLs (Figure 5A). In this analysis, we achieved a 100% separation of TAG and PL, indicated as a complete absence of 17:0 fatty acids derived from PL in the TAG sample and a corresponding complete absence of 13:0 fatty acids derived from TAG in the PL sample (Figure 5A). Thus SPE is highly efficient at separating lipid classes. This analysis does not indicate that all TAGs of different chain lengths will be purified equivalently by SPE and detected by GCMS with similar efficiency. It only assures that TAG and PL lipids in total are completely separated from each other.
A sample GC-MS trace of N2 wild type animals taken through solid phase extraction and FAME preparation is shown in Figure 5B. For both TAG and PL, peaks can be identified based upon retention time and parent and daughter ions on the mass spectrum, and the total area under the identified peaks normalized to the area of the respective internal standards. To determine the normalized amount of TAG, the area under all peaks of the TAG chromatogram, except the 13:0 standard, were added together and divided by the area under the 13:0 peak. Likewise, to determine the normalized total PL amount, the area under all peaks, except the 17:0 standard, were added together and divided by the area under the 17:0 peak. The normalized values for the TAG and PL samples are then used to calculate the final TAG/PL ratio for the sample:
<img src="/files/ftp_upload/50180/50180eq1.jpg" alt="Equation 1" fo:content-width="2.6in" fo:src="/files/ftp_upload/50180/50180eq1highres.jpg"/> 
It should be noted that the data from SPE-GCMS permit a much more sophisticated analysis of fatty acids present in each lipid fraction than simple calculation of the total TAG to PL ratio. For example, the investigator can integrate a single peak corresponding to a single fatty acid and compare its normalized abundance across samples (e.g. comparing in N2 wild-type versus daf-2 RNAi the integrated area of the 18:0 peak divided by the 13:0 standard peak, normalized to total PL mass divided by PL 17:0 standard peak area):
<img src="/files/ftp_upload/50180/50180eq2.jpg" alt="Equation 1" fo:content-width="4.873in" fo:src="/files/ftp_upload/50180/50180eq2highres.jpg"/> 
Should the investigator choose, it would also be possible to determine the percentage of any given fatty acid in the TAG or PL fractions as a percentage of the total amount of fatty acid quantitated (as an example 18:0 in the TAG fraction in N2 worms):
<img src="/files/ftp_upload/50180/50180eq3.jpg" alt="Equation 1" fo:content-width="4.123in" fo:src="/files/ftp_upload/50180/50180eq3highres.jpg"/>
<img src="/files/ftp_upload/50180/50180fig1.jpg" alt="Figure 1" fo:content-width="3.25in" fo:src="/files/ftp_upload/50180/50180fig1highres.jpg"/> 
 Figure 1. Typical workflow for the overall experiment. Day 1, frozen RNAi library plates are stamped onto Amp/Tet LB agar plates and incubated at 37 &deg;C. Day 2, Amp/Tet plates are used to seed liquid cultures of RNAi clones in deep well blocks. Day 3, bacteria are concentrated by centrifugation and transferred to 96-well RNAi plates. Day 4, L1 hatchling C. elegans are added to RNAi plates in liquid and allowed to dry. Day 7, animals are collected in liquid, stained with Nile red in 40% isopropanol, mounted on 96-well Teflon slides, and imaged with a high-throughput microscope.
<img src="/files/ftp_upload/50180/50180fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50180/50180fig2highres.jpg"/> 
 Figure 2. Fixative-based Nile red stains major C. elegans fat stores. Representative bright field and GFP fluorescence images are shown of N2 worms fed on fasn-1 (small, low fat), lpd-3 (low fat), control (empty vector), or daf-2 (high fat) RNAi clones. Samples were processed as per the protocol (schematic in Figure 1). Animals fixed, stained in Nile red, and imaged result in comparable lipid levels as obtained from biochemical lipid measurement. Isopropanol-fixed Nile red fluorescence levels, acquired from at least 6 biological replicates, are shown in the first row. Quantitative triglyceride levels, reflective of overall neutral lipid stores when normalized to overall phospholipid levels (TAG/PL), taken from 4 biological replicates, are shown in the second row. It is important to note that the absolute quantitation achieved from fixative Nile red staining and biochemical lipid determination often do not match perfectly. In this instance, though qualitatively similar, fasn-1(RNAi) has more different triglyceride mass vs. control by biochemical methods than indicated by microscopy, lpd-3 is comparably different, and daf-2(RNAi) is less different, though all differences are highly statistically significant and measurements were highly reproducible. Data shown are mean &plusmn; SEM (*, P &le;0.01; **, P &le; 0.0001 relative to control, determined by unpaired, two-tailed T-test assuming equal variance).
<img src="/files/ftp_upload/50180/50180fig3.jpg" alt="Figure 3"/> 
 Figure 3. Fat mass determined by fixation Nile red staining at different larval stages. Animals were grown on OP50 bacteria and collected at the L1, L2, L3, L4 and gravid adult developmental stages. Images were captured following fixation staining and analyzed using a customized Matlab script to determine the 90th percentile of pixel intensity over the identified worm area. Note that the lipid levels of animals greatly increase as the animal develops from L2 to L4 stage, and then decrease slightly as the animal begins to divert calories toward the proliferation of the germline in the adult stage. Data shown are mean &plusmn; SEM for 6-8 replicates (**, P &le; 0.0001 relative to gravid adult, determined by unpaired, two-tailed T-test).
<img src="/files/ftp_upload/50180/50180fig4.jpg" alt="Figure 4"/> 
 Figure 4. High reproducibility across independent biological replicates. Shown is a scatter plot of Nile red fluorescence intensities across biological replicates (n = 6-8). For each replicate, all values are normalized to the mean intensity of the empty vector controls. Data shown are mean &plusmn; SEM (**, P &le; 0.0001 relative to control, determined by unpaired, two-tailed T-test assuming equal variance).
<img src="/files/ftp_upload/50180/50180fig5.jpg" alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/50180/50180fig5highres.jpg"/> 
 Figure 5. GC-MS chromatograms of SPE separated standards and wild type TAG and PL abundance. (A) GC-MS traces are shown of the SPE separation of TAG and PL standards. Note the complete absence of 13:0 fatty acid in the PL trace and the corresponding absence of the 17:0 fatty acid in the TAG trace, indicating complete separation of TAG (tritridecanoin) from PL (1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). (B) A representative full spectrum of TAG and PL from a sample of N2 wild type animals fed vector control RNAi (HT115 bacteria with L4440 empty vector RNAi). One microliter of sample was injected into an Agilent 6890/5973N GCMS according to the protocol, and individual peaks were identified by retention time and their respective mass spectra. Abbreviations: cyclo, cyclopropane fatty acid; iso: iso-methyl branched chain fatty acid; GLA, gamma linolenic acid; ALA, alpha linolenic acid; DGLA, dihomo gamma linolenic acid; EPA, eicosapentaenoic acid. <a href="https://www.jove.com/files/ftp_upload/50180/50180fig5large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans at JoVE.com

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

biochemical-high-throughput-microscopic-assessment-fat-mass

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Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

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

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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. ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

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