This procedure aims to identify and measure prostaglandins and other eicosanoids in extracts of sea elegance. First, grow the worms in large scale cultures. Then using a liquid liquid extraction technique, extract the hydrophilic lipids from the worm tissue, inject the lipid extracts into an HPLC column coupled to a mass spectrometer.
Then analyze the data from the liquid chromatography tandem mass spectrometry. Ultimately this lc MSMS approach to purified warm extracts can identify new metabolites and also measure known metabolites such as the S series prostaglandins. So the main advantage of this technique over existing methods, such as enzyme immunoassays, is that it is highly sensitive and specific, while at the same time, comprehensive and flexible demonstrating this technique will be Hugh Hong, a graduate student from my laboratory For supplemental feeding of the worms pellet and NA 22.
Bacterial culture by centrifugation, discard the supernatant and resus suspend the bacterial pellet in 100 milliliters of M nine buffer. Store the concentrated bacterial solution at four degrees Celsius. Start the worm cultures on five extra large NGM plates.
Continue to propagate the worms for three to four generations until the plates are full of gravity. Adults to prevent starvation at approximately one milliliter of the concentrated bacteria to the worm plates as needed. Air dry completely before placing the lid.
Next, wash the grab hermaphrodites off the plates with M nine buffer. Harvest the worms in a 50 milliliter conical polypropylene tube. Then allow the worms to settle to the bottom.
Discard the supernatant containing bacteria eggs and larval stage worms. Now Resus, suspend the remaining worms in 15 milliliters of M nine buffer and gently mix. Distribute 200 microliters of the worm suspension to each of 60 seeded N GM plates.
Keep the worm solution mixed well until all plates have been seeded to ensure that they receive roughly equal numbers of worms. Disperse the worms across the plates. Grow the worms for two to four generations, supplementing with one milliliter of concentrated bacteria per plate per 12 to 24 hour period.
When the plates are full of GR adults, harvest the worms one strain at a time. Wash the worms off the plates with M nine buffer and transfer to six 50 milliliter polypropylene tubes. Fill the tubes with M nine buffer to wash the worms.
When the grab adult settle to the bottom, remove about 35 milliliters of the supernatant. Then pull the worms into one or two tubes. Repeat the M nine wash three times or until the supernatant is transparent with a large bore pasti pipette.
Transfer as many worms as possible to a 15 milliliter polypropylene conical tube. Centrifuge at 1000 RCF for five minutes and discard the supernatant. Repeat the harvesting, washing, and consolidating steps for the other strains.
Store stocks of the harvested worm strains at minus 80 degrees Celsius. Note the weight of a polypropylene conical tube using a red hot razor blade. Cut a frozen polypropylene tube containing harvested worms near the six milliliter mark.
Under sterile conditions. Transfer about six grams of frozen worm pellet to the thought sample. Add one nanogram of the internal standard and 12 milliliters of ice cold acetone saline solution.
With BHT mix, well distribute 1.5 milliliters of the worm slurry to 12 five milliliter self-standing plastic tubes to each tube. Add 0.7 milliliters of the serious stabilized zirconium oxide beads. Homogenize the worm suspension if necessary.
Repeat the process for another minute to obtain less than 10%of intact worms using a nine inch pasture pipette. Carefully transfer the homogenate but not the beads into four 10 milliliter conical glass tubes. Combine the beads from three tubes and wash with one milliliter of acetone saline solution containing BHT.
Repeat this wash for the remaining bead containing tubes. Transfer the wash solution to the glass tubes on ice. Next, centrifuge the homogenate at 1000 Gs for 10 minutes.
At four degrees Celsius, transfer the supernatant from each tube to a clean 10 milliliter glass conical tube. Then in a chemical hood, add equal volume of hexane to each tube vortex at maximum speed for 30 seconds. After centrifusion at four degrees Celsius at 1000 Gs for 10 minutes.
Discard the upper phase containing the hexane and neutrally charged lipids at two millimolar formic acid to acidify the lower aqueous phase. Testing with pH strips for a pH of 3.5. Now, add equal volumes of chloroform to each tube vortex at maximum speed for 30 seconds.
Centrifuge the samples at four degrees Celsius at about 1000 GS per 10 minutes. Insert a glass pipette tip into the lower chloroform phase and try to avoid the solid interface by tilting the tube. Pull the extracts from four tubes into a single 15 milliliter conical glass tube.
Purge the air in the glass tube with nitrogen gas and incubate at minus 20 degrees Celsius for 24 hours to two weeks. After thawing the extract, discard the remaining milky aqueous top layer if present. Using a new past pipette.
Transfer the chloroform to a labeled Teflon lined half DRM glass vial in a chemical hood. Evaporate the organic solvent in a gentle stream of nitrogen gas store samples at minus 20 degrees Celsius for up to two weeks. First, prepare stock solutions of individual reference prostaglandins in methanol.
Now generate a standard curve by serial dilution in 80%methanol. Next, dissolve the dried sea elegance lipid extracts in 200 microliters of 80%methanol for analysis. Inject 20 to 50 microliters of the sample into the reverse phase column and perform gradient analysis as detailed in the text protocol using negative ion mode ESI interface.
Introduce the column effluent into the mass spectrometer. Set the collision gas at 10 electron volts. The collision energy at minus 35 electron volts.
The temperature at 600 degrees Celsius. The decluttering potential at minus 90 and the cell exit potential at minus 11 generate a standard curve to analyze the different classes of prostaglandins present in the extracted samples using analyst software. Process the data to identify prostaglandins present in the worm extracts.
Survey scans of the wild type and fat three mutant worm extracts show the global levels of ions within the mass range of 315 to 360 atomic mass units. Compared to the wild type worms, the fat three mutants lack most 20 carbon pfas as exemplified by an F three class prostaglandin. With master charge ratio 3 51 highlighted in gray, MRM analyses can identify the different classes of prostaglandins.
For instance, the F1 class prostaglandins are present in wild type extracts as well as several other hydrophobic compounds with greater retention times varying the mass transition allows for further analysis of the F two and F three class prostaglandins indicating the worm extracts contain multiple prostaglandin isomers of each class. Comparison of the collision induced decomposition of the sea elegance prostaglandin to the standard prostaglandin can be used to map to product ions of expected cleavage sites. In order to prevent lipid oxidation.
Remember to take the appropriate precautions. For example, add the antioxidant VHT to the indicated solution and purge extract with nitrogen gas.
