Name: prostaglandin extraction and analysis in caenorhabditis elegans
Uploaded: 2013-06-25T13:00:00
Duration: 8 min 50 s
Description: Watch this Scientific Journal Video about Prostaglandin Extraction and Analysis in Caenorhabditis elegans at JoVE.com

We thank the UAB Targeted Metabolomics and Proteomics Laboratory, which has been supported in part by the UAB Skin Disease Research Center (P30 AR050948 to C. Elmets), the UAB-UCSD O'Brien Acute Kidney Injury Center (P30 DK079337 to A. Agarwal) and the UAB Lung Health Center (R01 HL114439, R01 HL110950 to J.E. Blalock). Support for the mass spectrometer was from a NCRR Shared Instrumentation grant (S10 RR19261 to S. Barnes). We also thank Ray Moore for technical support. C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH. This work was supported by the NIH (R01GM085105 to MAM, including an ARRA administrative supplement).

The protocol as described below is divided into three sections: worm culture, prostaglandin extraction, and prostaglandin analysis. As noted, there are multiple points when samples can be temporarily stored at -80 &deg;C or -20 &deg;C.
1. Worm Culture
We recommend approximately 6 g of mixed stage worms for comprehensive LC-MS/MS. This should provide enough material for detection at least six separate injections. For quantification of known PGs by MRM in a single injection or two, 1-2 g are sufficient. Analysis of extracts from synchronized cultures consisting of a specific stage is also possible. Up to 4 different strains can be grown simultaneously. For example, a wild type control and three different mutant strains.
<ol>
	<li>Prepare 65 X-large (16 cm) NGM plates and concentrated bacteria for supplemental feeding. Use NA22 bacteria for seeding. To make concentrated bacteria for supplemental feeding, grow at least four liters of NA22 in LB medium for 16-24 hr. Spin down, remove the supernatant, and resuspend bacteria in 100 ml of M9 buffer. 200 to 400 ml of this concentrated bacterial solution may be required for each worm strain. Store at 4 &deg;C.</li>
	<li>Start worm cultures on 5 X-large plates. Grow worms for approximately 3 to 4 generations until plates are full of gravid adults. Concentrated bacteria should be added to plates to prevent starvation, as necessary (~ 1 ml/plate and let dry completely before replacing lid).</li>
	<li>Wash gravid hermaphrodites off plates with M9 buffer and collect the worms in a 50 ml conical polypropylene tube.</li>
	<li>Allow gravid hermaphrodites to settle to the bottom (usually about 3-5 min). Remove supernatant, including bacteria, eggs, and larval stage worms. Resuspend in 15 ml M9 buffer and gently mix.</li>
	<li>Transfer 200 &mu;l of worm suspension to each of 60 seeded NGM plates. Disperse worms across the plates. Keep worm solution mixed well until all plates have been seeded to ensure that they receive roughly equal numbers of worms.</li>
	<li>Supplement with concentrated bacteria as needed (~1 ml/plate/12 to 24 hr period) to prevent starvation. Allow plates to air dry before replacing lids. Grow worms for 2-4 generations, depending on the original number of seeded worms, or until plates are full of gravid adults.</li>
	<li>Harvest worm plates one strain at a time. Wash worms off the plates with M9 buffer and collect in 50 ml polypropylene tubes (e.g. 6 tubes). Use M9 buffer to wash a plate, then transfer the worm-filled buffer to the next plate. After washing a number (e.g. 10th) of plates, transfer the worm-filled buffer to the 50 ml tube. Fill the tube to the top with M9 buffer. Repeat the wash for the rest of the plates.</li>
	<li>As the gravid adults settle to the bottom in 5-6 min, remove the supernatant (~ 35 ml) and consolidate into one or two tubes. Fill the tubes to 50 ml with fresh M9 buffer, let stand for 3-5 min, and remove supernatant leaving gravid adults. Repeat 3 times or until the supernatant is transparent.</li>
	<li>Using a large bore Pasteur pipette, transfer as many worms as possible to two 15 ml polypropylene conical tubes. Spin down at 1,000 rcf (~3,500 rpm in CentraSL2) for 5 min in a clinical centrifuge. Remove supernatant and repeat until all worms are transferred and pelleted in the same tube. Remove as much supernatant as possible and store worms at -80 &deg;C. Repeat for all strains.</li>
</ol>
2. Prostaglandin Extraction
The reagents needed for extraction are shown below. The method is modified from that of Golovko and Murphy17 and includes acetone extraction followed by liquid-liquid purification, which enhances LC-MS/MS sensitivity. A Bullet Blender 5 homogenizer is used in the procedure, although similar results can be obtained with a Dounce homogenizer. Butylated hydroxytoluene (BHT) and evaporation under Nitrogen (N2) gas are used to prevent oxidation. All glassware should be siliconized (Sigmacote) to reduce lipid binding. Extraction with organic solvents should be performed in a chemical hood.
<ol>
	<li>Weigh a 50 ml polypropylene conical tube. Transfer approximately 6.0 g of frozen worms from the 15 ml conical tube stored at -80 &deg;C by cutting through the tube with a hot razor blade near the 6.5 ml mark. A methanol-cleaned spatula can be used to remove the frozen worm pellet from the bottom of the tube. By cutting through the pellet to retrieve the lower portion, less dense larval worms and residual bacteria from the top of the pellet are left behind.</li>
	<li>Transfer the frozen worms to the pre-weighed 50 ml conical tube. If multiple samples are being processed, use the same amount (6.0 g) of tissue for comparative studies. As the worms thaw into a paste, add or remove worms with the spatula to obtain the desired mass. Optional: add 1.00 ng of PGF2&alpha;-d4 standard as an internal control for extraction efficiency.</li>
	<li>Add 12 ml of ice-cold 2:1 acetone/saline with 0.005% BHT to the worm suspension and mix well by vortexing. Dispense 1.5 ml of the worm slurry to each of twelve 5 ml self-standing plastic tubes for use in the Bullet Blender.</li>
	<li>Add 0.7-0.8 ml of 0.5 mm diameter Ceria stabilized zirconium oxide beads to each 5 ml tube. Close caps tightly and load the 12 tubes into the Bullet Blender 5 homogenizer. Run the blender for 3 min at speed 8-9 and place the tubes on ice. Be sure to close the caps very tightly or the content may spill out. Check 10 &mu;l of homogenate from one tube on a slide using a stereoscope. There should be very few intact worms remaining. Repeat at the same speed for another minute, if necessary.</li>
	<li>Evenly transfer the homogenates to four 10 ml conical glass tubes using a 9" Pasteur pipette. Avoid transferring beads. Wash the beads from three tubes sequentially with 1 ml of 1:2 acetone/saline containing BHT. Repeat for other tubes until all beads are washed. Transfer the remaining solution (~ 4 ml) to the 10 ml glass tubes and place them on ice.</li>
	<li>Centrifuge the 10 ml glass tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min. Transfer the supernatant from each tube to a clean 10 ml glass conical tube.</li>
	<li>In a chemical hood, add an equal volume of hexane to each 10 ml glass tube. For example, add 3.5 ml hexane to 3.5 ml acetone/saline solution. Vortex at maximum speed for 30 sec.</li>
	<li>Centrifuge the 10 ml glass tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min. Discard the upper phase containing hexane and neutrally charged lipids.</li>
	<li>Acidify the lower phase to pH 3.5 using 2 M formic acid (approximately 100 - 150 &mu;l). Test pH using pH strips.</li>
	<li>Add an equal volume of chloroform to each tube. Vortex at maximum speed for 30 sec and centrifuge the tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min.</li>
	<li>Remove and discard the upper aqueous phase. Insert a pipette tip through any residual milky interphase to the lower chloroform phase, avoiding the interphase. Collect the lower phase from each of the 4 tubes to a clean 15 ml conical glass tube. Incubate at -20 &deg;C for at least 24 hr. At this point, the extract can be stored at -20 &deg;C for up to two weeks.</li>
	<li>Take the extract out of the freezer and discard the remaining milky aqueous top layer, if present. Transfer the chloroform phase to a labeled Teflon-lined &frac12;-Dram glass vial using a new Pasteur pipet. In a chemical hood, evaporate the chloroform soluble fraction to dryness under a gentle flow of N2 gas. The dried extract can be stored at -20 &deg;C for up to 2 weeks. An overall scheme used for PG extraction and analysis is shown in Figure 1.</li>
</ol>
3. Prostaglandin Analysis
<ol>
	<li>Prepare stock solutions of individual reference prostaglandins, such as PGF2&alpha; or PGF2 &alpha;-d4 (1 &mu;g/ml) in methanol and dilute with methanol:water (8:2 v/v) to obtain a working solution. Prepare a series of dilutions (100, 10, 1, 0.1, 0.01 ng/ml) to generate a standard curve.</li>
	<li>Add 200 &mu;l methanol:water (8:2 v/v) to the dried C. elegans lipid extract(s) and mix thoroughly. If less than 6 g of worm tissue was extracted, the dried extract should be resuspended in less volume of methanol:water solution.</li>
	<li>Prepare the mobile phase consisting of 0.1% formic acid in water [A] and acetonitrile containing 0.1% formic acid [B].</li>
	<li>Set up the autosampler at 4 &deg;C and place the sample vials in a 70-count 1.5 ml vial rack.</li>
	<li>Equilibrate the Synergy hydro RP-C18 column with 0.1% formic acid at a flow rate of 0.2 ml/min for about 5 min.</li>
	<li>Inject the sample (20-50 &mu;l) into the column. Perform gradient elution starting with 10% B and going up to 80% B from 0-11 min, 80-100% B from 11-14 min, and returning back to 10% B at 16 min. The total run time is 20 min. The retention times of 13 authentic standards are shown in Table 1 for reference3.</li>
	<li>Introduce the column effluent into the mass spectrometer using an ESI interface operating in the negative ion mode. Nitrogen is used as a nebulizer and curtain gas (CUR = 10). The collision gas, collision energy, and temperature are set at 10, -35 eV and 600 &deg;C, respectively. Declustering potential (DP), collision energy (CE), and cell exit potential (CXP) are set at -90, -35 and -10, respectively.</li>
	<li>For survey scans (Q1 scans), use negative ion mode in the mass range of m/z 315-360 for most PGs (Figure 2). The range can be altered, depending on the mass of the compound(s) of interest. Extract ions from total ion current (TIC) of LC-MS ion chromatograms and determine whether the extracted ions correspond to deprotonated or adduct ions, or to fragment ions.</li>
	<li>For MRM experiments, mass transition m/z 355/311 is used to detect the F1 class, m/z 353/193 is used for the F2 class, and m/z 351/193 or 351/191 is used for the F3 class (Figure 3). MRM is also used to detect the internal standard (for example, m/z 357/197 for PGF2&alpha;-d4) and to generate the standard curve. See Murphy et al. for additional information on PG mass transitions18.</li>
	<li>For MS/MS experiments, use m/z 355 for F1 class, m/z 353for F2 class, and m/z 351for F3 class PGs. C. elegans MS/MS data for F-series PGs is available in Figure 4 and Table 23,16. MS/MS data for mammalian eicosanoids is available at <a href="http://www.lipidmaps.org" target="_blank">www.lipidmaps.org</a>.</li>
	<li>Process the analytical data using Analyst software(Version 1.4.2, Applied Biosystems).</li>
</ol>

<ol>
	<li>Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+synthesis+and+adiposity+regulation+in+Caenorhabditis+elegans.">Fat synthesis and adiposity regulation in Caenorhabditis elegans.</a> Trends Endocrinol. Metab. 20, 58-65 (2009).</li><li>Marza, E., Lesa, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyunsaturated+fatty+acids+and+neurotransmission+in+Caenorhabditis+elegans.">Polyunsaturated fatty acids and neurotransmission in Caenorhabditis elegans.</a> Biochem. Soc. Trans. 34, 77-80 (2006).</li><li>Edmonds, J. W., 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=Insulin/FOXO+signaling+regulates+ovarian+prostaglandins+critical+for+reproduction.">Insulin/FOXO signaling regulates ovarian prostaglandins critical for reproduction.</a> Dev. Cell. 19, 858-871 (2010).</li><li>Kniazeva, M., Shen, H., Euler, T., Wang, C., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+maternal+phospholipid+composition+and+IP(3)-dependent+embryonic+membrane+dynamics+by+a+specific+fatty+acid+metabolic+event+in+C.+elegans.">Regulation of maternal phospholipid composition and IP(3)-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans.</a> Genes Dev. 26 (3), 554-566 (2012).</li><li>Kosel, 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=Eicosanoid+formation+by+a+cytochrome+P450+isoform+expressed+in+the+pharynx+of+Caenorhabditis+elegans.">Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans.</a> Biochem. J. 435, 689-700 (2011).</li><li>Kubagawa, H. 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=Oocyte+signals+derived+from+polyunsaturated+fatty+acids+control+sperm+recruitment+in+vivo.">Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo.</a> Nat Cell Biol. 8, 1143-1148 (2006).</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=N-acylethanolamine+signalling+mediates+the+effect+of+diet+on+lifespan+in+Caenorhabditis+elegans.">N-acylethanolamine signalling mediates the effect of diet on lifespan in Caenorhabditis elegans.</a> Nature. 473, 226-229 (2011).</li><li>Gerisch, 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+bile+acid-like+steroid+modulates+Caenorhabditis+elegans+lifespan+through+nuclear+receptor+signaling.">A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 5014-5019 (2007).</li><li>Edison, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+pheromones+regulate+multiple+complex+behaviors.">Caenorhabditis elegans pheromones regulate multiple complex behaviors.</a> Curr. Opin. Neurobiol. 19, 378-388 (2009).</li><li>Funk, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandins+and+leukotrienes:+advances+in+eicosanoid+biology.">Prostaglandins and leukotrienes: advances in eicosanoid biology.</a> Science. 294, 1871-1875 (2001).</li><li>Wang, D., Dubois, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eicosanoids+and+cancer.">Eicosanoids and cancer.</a> Nat. Rev. Cancer. 10, 181-193 (2010).</li><li>Cha, Y. I., Solnica-Krezel, L., DuBois, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+prostanoids:+deciphering+the+developmental+functions+of+cyclooxygenase-derived+prostaglandins.">Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins.</a> Dev. Biol. 289, 263-272 (2006).</li><li>Hata, A. N., Breyer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+and+signaling+of+prostaglandin+receptors:+multiple+roles+in+inflammation+and+immune+modulation.">Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation.</a> Pharmacol. Ther. 103, 147-166 (2004).</li><li>Sugimoto, Y., Narumiya, S., Ichikawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+function+of+prostanoid+receptors:+studies+from+knockout+mice.">Distribution and function of prostanoid receptors: studies from knockout mice.</a> Prog. Lipid Res. 39, 289-314 (2000).</li><li>Basu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoprostanes:+novel+bioactive+products+of+lipid+peroxidation.">Isoprostanes: novel bioactive products of lipid peroxidation.</a> Free Radic. Res. 38, 105-122 (2004).</li><li>Hoang, H. D., Prasain, J. K., Dorand, D., Miller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterogenous+mixture+of+F-series+prostaglandins+promotes+sperm+guidance+in+the+C.+elegans+reproductive+tract.">A heterogenous mixture of F-series prostaglandins promotes sperm guidance in the C. elegans reproductive tract.</a> PLoS Genetics. 9 (1), e1003271 (2013).</li><li>Golovko, M. Y., Murphy, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+LC-MS/MS+procedure+for+brain+prostanoid+analysis+using+brain+fixation+with+head-focused+microwave+irradiation+and+liquid-liquid+extraction.">An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction.</a> J. Lipid Res. 49, 893-902 (2008).</li><li>Murphy, R. 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=Electrospray+ionization+and+tandem+mass+spectrometry+of+eicosanoids.">Electrospray ionization and tandem mass spectrometry of eicosanoids.</a> Anal. Biochem. 346, 1-42 (2005).</li><li>Milne, G. L., Yin, H., Morrow, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+biochemistry+of+the+isoprostane+pathway.">Human biochemistry of the isoprostane pathway.</a> J. Biol. Chem. 283, 15533-15537 (2008).</li><li>Lehtonen, M., Reisner, K., Auriola, S., Wong, G., Callaway, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometric+identification+of+anandamide+and+2-arachidonoylglycerol+in+nematodes.">Mass-spectrometric identification of anandamide and 2-arachidonoylglycerol in nematodes.</a> Chem. Biodivers. 5, 2431-2441 (2008).</li><li>Kulas, J., Schmidt, C., Rothe, M., Schunck, W. H., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450-dependent+metabolism+of+eicosapentaenoic+acid+in+the+nematode+Caenorhabditis+elegans.">Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans.</a> Arch. Biochem. Biophys. 472, 65-75 (2008).</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></ol>

The authors have no conflicts of interest.

We describe a procedure for eicosanoid extraction and analysis, focusing on F-series PGs. There are several parts of the procedure that can be problematic. First, it is critical that the worm cultures do not starve, as starvation can alter PG metabolism. For supplemental feeding, NA22 bacteria are recommended instead of the more commonly used OP50 bacteria because NA22 reaches higher density. However, the NA22 strain lacks antibiotic resistance and is more susceptible to contamination. Second, worms synthesize individual PG isomers in low abundance relative to mammalian tissues. This may be due in part to redundancy among the numerous isomers. We recommend about 6 g of tissue for comprehensive analysis and stronger signals. Less tissue (1-2 g) can be used if the dried extract is resuspended in less methanol:water solution to increase PG concentration. However, only one to two injections can be performed. The detection limit of our LC-MS/MS system is about 10 pg PGF2&alpha; /ml. One ml of densely packed mixed stage worms (about 1 g) yields roughly 25-50 pg of CePGF2. More sensitive mass spectrometry systems can reduce the amount of worm tissue required for analysis. Third, differences in PG extraction efficiency among samples can cause variable results. We have found that extraction efficiency is very similar when tissues are extracted in parallel. To determine the efficiency, add 1.0 ng of PGF2&alpha;-d4 at the homogenization step as an internal control. MRM using mass transition m/z 357/197 is used to measure PGF2&alpha;-d4 concentration relative to a 1 ng/ml standard solution. The amount of PGF2&alpha;-d4 lost during extraction is then calculated.
The chromatography parameters and mass transitions we include can be altered to detect other PGs and eicosanoids. Despite considerable effort, we have not been able to identify D-series or E-series PGs in the extracts17. Furthermore, we have not detected 8-iso PGF2&alpha; and 8-iso PGE2 that are characteristic of free radical-initiated peroxidation, which generates a nonselective mixture of PG stereoisomers19. Whether worms synthesize other PG types is not known. Endocannabinoids and various epoxy and hydroxy metabolites of arachidonic and eicosapentaenoic acids have been found in C. elegans extracts5,7,20,21. We recommend the use of both MRM and MS/MS compared to authentic standards for quantification and identification, respectively. For novel eicosanoids, these analyses should be combined with a comparative study of fat mutants, which are deficient in PUFA synthesizing enzymes22, as well as a functional assay, if possible. A caveat to analyzing fat mutants is that minute quantities of PUFAs present in worms are sufficient for PG synthesis. For example, fat-2(wa17) mutants have a small amount of D12 desaturase activity (~5% of wild type22) and these mutants still produce PGs6. fat-3(wa22) and fat-4(wa14) mutants fail to synthesize PGs derived from arachidonic and eicosapentaenoic acids16. We also have found that fat mutants compensate for the loss of PUFA classes by up-regulating PG synthesis from remaining classes. For example, fat-3(wa22) mutants fail to synthesize most PGs derived from 20-carbon PUFAs. Instead, they appear to up-regulate novel PGs derived from 18-carbon PUFAs16. To date, we have not identified a C. elegans strain that is completely deficient in PG synthesis. It is possible that PGs are essential for growth or development.

Prostaglandins (PGs) are an important class of lipid hormones that have been extensively investigated and implicated in regulating reproduction, immunity, and development in a wide array of organisms12-14. PGs are ideally suited for comprehensive systems biology analyses because they comprise a series of lipids derived from a common precursor(s). The nematode C. elegans is one of the most widely used model organisms to address fundamental questions in genetics and systems biology.
We have demonstrated that C. elegans synthesizes F-series PGs that guide motile sperm to oocytes3,6,16. F-series PGs derived from 20-carbon PUFAs with three, four, and five double bonds, comprising the F1, F2, and F3 classes, respectively have been identified3,16. These PGs are synthesized independent of cyclooxygenase enzymes, yet PGF1&alpha; and PGF2&alpha; stereoisomers are still generated. For qualitative and quantitative analyses of C. elegans PGs, LC-MS/MS is a powerful and sensitive analytical technique. Before performing this analysis, it is important to develop an optimized extraction method because the performance of the analytical process heavily depends on the extract quality. Liquid chromatography and mass spectrometry can only provide anticipated mass to charge ratio (m/z), as well as desirable peak shape and retention time of the PG parent ion. Metabolic profiling of PGs in C. elegans is a challenging task requiring various MS acquisition strategies.
LC-MS full scan or survey scanning (Q1 scanning) has the advantage of ensuring that most ionizable PGs will generate a mass spectrometric response. While the survey scan provides useful information on total profiling of PGs with respect to wild-type and mutant C. elegans, detection of minor PGs is compromised due to low sensitivity. Nevertheless, LC-MS survey scanning can give a global view of PGs and is particularly useful for analyzing mutants predicted to affect metabolism of a large PG population.
In metabolite identification, LC-MS/MS analysis of chemically synthesized PG standards is first performed to obtain their product ion spectra as reference compounds. These spectra can be compared to the spectrum of an unknown metabolite. Multiple reaction monitoring (MRM) mode is used for enhanced sensitivity and specificity. An MRM experiment is accomplished by specifying the parent ion/product ion mass transition of a compound. By knowing the mass and structure of the target analytes, it is possible to predict theoretical MRM transitions for many unknown metabolites.
An optimized LC-MS/MS method for profiling isomeric PGs in C. elegans has not been reported. Here we describe an extraction and integrated mass spectrometry approach consisting of LC-MS and LC-MS/MS methods for detecting, quantifying, and investigating C. elegans PG metabolites. This approach can be applied to other eicosanoids.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>X-large (16 cm) plates</td>
			<td>Fisher Scientific</td>
			<td>0875714</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E. coli strain NA22</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>NA22</td>
			<td><a href="http://www.cgc.cbs.umn.edu" target="_blank">http://www.cgc.cbs.umn.edu</a></td>
		</tr>
		<tr>
			<td>Acetone, 399.5%</td>
			<td>Fisher Scientific</td>
			<td>A928-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Butylated Hydroxytoluene</td>
			<td>MP Biomedicals</td>
			<td>101162</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Fisher Scientific</td>
			<td>H302-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid, 399%</td>
			<td>Acros</td>
			<td>AC27048-0010</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>Chloroform, HPLC grade</td>
			<td>Acros</td>
			<td>AC26832-0010</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>PGF2&alpha;-d4</td>
			<td>Cayman Chemicals</td>
			<td>316010</td>
			<td>Cayman has a wide array of standards available for purchase</td>
		</tr>
		<tr>
			<td>Sterile screw cap self-standing 5 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-222-651</td>
			<td>For use with Bullet Blender</td>
		</tr>
		<tr>
			<td>0.5 mm zirconium oxide beads</td>
			<td>Next &gt;&gt;&gt; Advance</td>
			<td>ZrOB05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml glass conical heavy-duty graduated centrifuge tubes</td>
			<td>Kimble Kimax</td>
			<td>45200-10</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>15 ml glass conical tubes</td>
			<td>Corning Pyrex</td>
			<td>8082-15</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sigmacote (Siliconizing reagent)</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon lined capped 1/2 Dram glass vial</td>
			<td>Fisher Scientific</td>
			<td>V1235C-TFE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CentraSL2 Clinical Centrifuge</td>
			<td>Thomas Scientific</td>
			<td>2517N92-TS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bullet Blender 5 blue homogenizer</td>
			<td>Next &gt;&gt;&gt; Advance</td>
			<td>BBY5MB</td>
			<td>A 40 ml Dounce homogenizer can be used as an alternative</td>
		</tr>
		<tr>
			<td>Synergi 4 &mu;m Hydro-RP 80 &Aring; LC Column 250 x 2.0 mm</td>
			<td>Phenomenenex</td>
			<td>00G-4375-B0</td>
			<td>HPLC Column</td>
		</tr>
		<tr>
			<td>SecurityGuard Cartridges AQ C18 4 x 2.0 mm</td>
			<td>Phenomenenex</td>
			<td>AJ0-7510</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SecurityGuard Guard Cartridges Kit</td>
			<td>Phenomenenex</td>
			<td>KJ0-4282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shimadzu Prominence HPLC with refrigerated auto sampler</td>
			<td>Shimadzu Scientific Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>Any standard HPLC system coupled to a triple quadrupole mass spectrometer should be sufficient</td>
		</tr>
		<tr>
			<td>API 4000 triple quadrupole mass spectrometer</td>
			<td>Applied Biosystems/MDS SCIEX</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">M9 buffer recipe (4 liter)</td>
					</tr>
					<tr>
						<td>12 g</td>
						<td>KH2PO4</td>
					</tr>
					<tr>
						<td>24 g</td>
						<td>Na2HPO4</td>
					</tr>
					<tr>
						<td>20 g</td>
						<td>NaCl</td>
					</tr>
					<tr>
						<td>Up to 4 liter</td>
						<td>Deionized water</td>
					</tr>
					<tr>
						<td colspan="2">Aliquot to eight 1 liter bottles. Autoclave and cool.</td>
					</tr>
					<tr>
						<td>0.5 ml/bottle</td>
						<td>1 M MgSO4</td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

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.

Sample preparation was carried out by liquid-liquid extraction adapted from Golovko and Murphy17. It provided excellent recovery of the internal standard (PGF2&alpha;-d4). The general scheme for PG extraction from C. elegans is shown in Figure 1. Chromatographic conditions were optimized to provide baseline separation of &gt;30 eicosanoid standards (Table 1 shows 13 standards). A Hydro-Rp column (250 x 2.0 mm i.d) with water and acetonitrile containing 0.1% formic acid provided the best separation and sensitivity. Survey scans of wild-type and fat-3 mutant extracts shown in Figure 2 document global levels of ions within the mass range of 315-360 atomic mass units. fat-3(wa22) mutants lack most 20-carbon PUFAs. An F3 class PG is highlighted. In Figure 3, MRM analyses of a wild-type extract show multiple PG isomers of the F1 (Figure 3A), F2 (Figure 3B), and F3 (Figures 3C and 3D) classes. The F3 class can be detected with either of two mass transitions, m/z 351/193 or m/z 351/191. CePGF2 is predominantly comprised of the PGF2&alpha;enantiomer. CePGF1 is likely to be PGF1&alpha; or its enantiomer. Figure 4 shows the collision-induced decomposition of CePGF2 (RT=11.8) compared to the PGF2&alpha; standard (RT=11.8). Minor differences in product ions are likely due to low CePGF2 abundance and co-eluting compounds in the extract.
<img alt="Figure 1" src="/files/ftp_upload/50447/50447fig1.jpg" /> 
 Figure 1. Schematic diagram of C. elegans PGs extraction and LC-MS analyses. Note that acetone/saline and chloroform both have 0.005% BHT to minimize lipid oxidation. Refer to the text for more details. <a href="https://www.jove.com/files/ftp_upload/50447/50447fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="4.75in" fo:src="/files/ftp_upload/50447/50447fig2highres.jpg" src="/files/ftp_upload/50447/50447fig2.jpg" /> 
 Figure 2. Survey scans of m/z 315-360 of wild-type and fat-3(wa22) mutant extracts. Liquid chromatography retention time (RT) is shown in panel [A]. Extracted ions at RT = 11.3 are shown for wild-type [B] and fat-3(wa22) [C] extracts. An F3 class PG with mass m/z 351 is highlighted. Cps, counts/sec; a.m.u., atomic mass unit.
<img alt="Figure 3" fo:content-width="4.25in" fo:src="/files/ftp_upload/50447/50447fig3highres.jpg" src="/files/ftp_upload/50447/50447fig3.jpg" /> 
 Figure 3. MRM analyses of wild-type extracts. F1 class PGs are detected with mass transition m/z 355/311 [A]. Notice that several hydrophobic compounds (RT &gt; 14 min), which are unlikely to be PGs, are also detected with this transition. F2 class PGs are detected with mass transition m/z 353/193 [B]. F3 class PGs are detected with either mass transition m/z 351/193 [C] or m/z 351/191 [D]. Notice that the extracts contain multiple PG isomers of each class. Numbers correspond to PGs shown in Table 2. The concentration of CePGF2 is 1.8 ng/ml. RT, retention time; cps, counts/sec.
<img alt="Figure 4" fo:content-width="5.5in" fo:src="/files/ftp_upload/50447/50447fig4highres.jpg" src="/files/ftp_upload/50447/50447fig4.jpg" /> 
 Figure 4. Collision-induced decomposition of chemically synthesized PGF2&alpha; and CePGF2. Arrows in panel [A] indicate product or fragmentation ions shared between PGF2&alpha; and CePGF2 (RT=11.8). The product ions at m/z 309 and m/z 193 are generated from indicated cleavage sites (highlighted a and b), correspond to the structures shown, and are characteristic of F-series PGs [B]. Cps, counts/sec. <a href="https://www.jove.com/files/ftp_upload/50447/50447fig4large.jpg" target="_blank">Click here to view larger figure</a>.
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Standard</td>
			<td>RT (min)</td>
			<td>[M-H]- m/z</td>
			<td>Key product ions in MS/MS</td>
		</tr>
		<tr>
			<td>20-hydroxy PGE2</td>
			<td>9.43</td>
			<td>367</td>
			<td>349, 331, 287, 234, 189, 129, 109</td>
		</tr>
		<tr>
			<td>PGF3-</td>
			<td>11.26</td>
			<td>351</td>
			<td>333, 307, 289, 271, 245, 219, 209, 193, 191, 171, 165, 111</td>
		</tr>
		<tr>
			<td>Thromboxane B2</td>
			<td>11.66</td>
			<td>369</td>
			<td>289, 191, 177, 169, 151</td>
		</tr>
		<tr>
			<td>PGF2-</td>
			<td>11.80</td>
			<td>353</td>
			<td>335, 309, 291, 273, 263, 247, 235, 209, 193, 171, 165, 111</td>
		</tr>
		<tr>
			<td>PGF1-</td>
			<td>11.79</td>
			<td>355</td>
			<td>337, 319,311, 301, 293, 275, 265, 237, 211, 195</td>
		</tr>
		<tr>
			<td>Lipoxin B4</td>
			<td>12.38</td>
			<td>351</td>
			<td>201, 191,189, 165, 155, 115, 107, 71, 59</td>
		</tr>
		<tr>
			<td>PGD2</td>
			<td>12.56</td>
			<td>351</td>
			<td>315, 271,203, 189</td>
		</tr>
		<tr>
			<td>5(S), 6(R)- Lipoxin A4</td>
			<td>12.83</td>
			<td>351</td>
			<td>235, 217, 189, 144, 135, 115, 99, 59</td>
		</tr>
		<tr>
			<td>PGA2</td>
			<td>14.02</td>
			<td>333</td>
			<td>315, 297, 271, 235, 191, 189, 175, 163, 137, 113, 109</td>
		</tr>
		<tr>
			<td>&Delta;12-PGJ2</td>
			<td>14.20</td>
			<td>333</td>
			<td>271, 189, 123</td>
		</tr>
		<tr>
			<td>Leukotriene B4</td>
			<td>14.73</td>
			<td>335</td>
			<td>181, 109, 93, 71, 69, 59, 57</td>
		</tr>
		<tr>
			<td>15d-&Delta;12,14-PGJ2</td>
			<td>16.80</td>
			<td>315</td>
			<td>297, 271, 217, 203, 158</td>
		</tr>
		<tr>
			<td>5(S)-HpETE</td>
			<td>17.88</td>
			<td>335</td>
			<td>97, 83, 81, 57</td>
		</tr>
	</tbody>
</table>
Table 1. Retention times and key product ions of 13 eicosanoid standards from the LC-MS/MS method3 . Over 30 standards were used to develop the LC-MS/MS program. The chromatographic retention times (RTs) differ, even among very similar PGs. An exception is PGF1&alpha; and PGF2&alpha;. Nevertheless, these PGs are distinguished by their parent ion masses ([M-H]- m/z) and collision-induced decomposition spectra (MS/MS).
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Prostaglandin class</td>
			<td>No. in Figure 3</td>
			<td>[M-H]- m/z</td>
			<td>Key product ions in MS/MS</td>
		</tr>
		<tr>
			<td>F1 (CePGF1)</td>
			<td>1</td>
			<td>355</td>
			<td>319, 301, 311, 293, 275, 265, 237, 223, 211, 195, 157</td>
		</tr>
		<tr>
			<td>F1</td>
			<td>2</td>
			<td>355</td>
			<td>311, 301, 293, 275, 265, 211, 195, 167, 157</td>
		</tr>
		<tr>
			<td>F2 (CePGF2)</td>
			<td>3</td>
			<td>353</td>
			<td>309, 291, 273, 263, 247, 209, 193, 171, 165, 127</td>
		</tr>
		<tr>
			<td>F2</td>
			<td>4</td>
			<td>353</td>
			<td>309, 291, 273, 263, 255, 247, 219, 209, 193, 171, 113</td>
		</tr>
		<tr>
			<td>F3</td>
			<td>5</td>
			<td>351</td>
			<td>315, 307, 289, 275, 249, 205, 193, 191, 167, 153, 139</td>
		</tr>
		<tr>
			<td>F3</td>
			<td>6</td>
			<td>351</td>
			<td>333, 289, 271, 261, 245, 223, 193, 191, 163</td>
		</tr>
	</tbody>
</table>
Table 2. Collision-induced decomposition product ions for several major C. elegans F1, F2, and F3 PGs shown in Figure 3. MS/MS of other less abundant isomers are not shown. These data are from multiple analyses and not all product ions may be visible in a given run. Given the complexity of the extracts, some less abundant product ions may derive from another parent ion with similar retention time or result from interference. Peak 2 is likely a stereoisomer of CePGF1 and peak 4 is likely a stereoisomer of CePGF2. See Edmonds, et al. (2010) for additional data3.

Watch this Scientific Journal Video about Prostaglandin Extraction and Analysis in Caenorhabditis elegans at JoVE.com

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.

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, ...

prostaglandin-extraction-and-analysis-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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 ...

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 ...

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...