Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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 °C or -20 °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.

1. 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 °C.
2. 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).
3. Wash gravid hermaphrodites off plates with M9 buffer and collect the worms in a 50 ml conical polypropylene tube.
4. 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.
5. Transfer 200 μl 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.
6. 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.
7. 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. 10^th) 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.
8. 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.
9. 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 °C. Repeat for all strains.

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.

1. Weigh a 50 ml polypropylene conical tube. Transfer approximately 6.0 g of frozen worms from the 15 ml conical tube stored at -80 °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.
2. 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 PGF_2α-d4 standard as an internal control for extraction efficiency.
3. 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.
4. 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 μl 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.
5. 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.
6. Centrifuge the 10 ml glass tubes at 4 °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.
7. 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.
8. Centrifuge the 10 ml glass tubes at 4 °C in a clinical centrifuge at ~1,000 x g for 10 min. Discard the upper phase containing hexane and neutrally charged lipids.
9. Acidify the lower phase to pH 3.5 using 2 M formic acid (approximately 100 – 150 μl). Test pH using pH strips.
10. Add an equal volume of chloroform to each tube. Vortex at maximum speed for 30 sec and centrifuge the tubes at 4 °C in a clinical centrifuge at ~1,000 x g for 10 min.
11. 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 °C for at least 24 hr. At this point, the extract can be stored at -20 °C for up to two weeks.
12. 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 ½-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 °C for up to 2 weeks. An overall scheme used for PG extraction and analysis is shown in Figure 1.

3. Prostaglandin Analysis

1. Prepare stock solutions of individual reference prostaglandins, such as PGF_2α or PGF_{2 α}-d4 (1 μg/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.
2. Add 200 μl 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.
3. Prepare the mobile phase consisting of 0.1% formic acid in water [A] and acetonitrile containing 0.1% formic acid [B].
4. Set up the autosampler at 4 °C and place the sample vials in a 70-count 1.5 ml vial rack.
5. 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.
6. Inject the sample (20-50 μl) 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.
7. 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 °C, respectively. Declustering potential (DP), collision energy (CE), and cell exit potential (CXP) are set at -90, -35 and -10, respectively.
8. 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.
9. 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 PGF_2α-d4) and to generate the standard curve. See Murphy et al. for additional information on PG mass transitions¹⁸.
10. 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 2^3,16. MS/MS data for mammalian eicosanoids is available at www.lipidmaps.org.
11. Process the analytical data using Analyst software(Version 1.4.2, Applied Biosystems).