The overall goal of this procedure is to measure the lifespan of sea Elgan worms that were treated with drugs of interest. This is achieved by first preparing OP 50, A bacterial strain, which is used to feed the worms. An age synchronized population of worms is then generated and seeded into 96 well plates together with the feeding bacteria OP 50.
Over the next two days, the worms grow to the L four larval stage and the drug FUDR is added to prevent reproduction. The following day, the worms reach adulthood and the drugs of interest are added to the individual wells. From this point forward, the number of living and dead worms is determined every two to three days until all worms have died.
Observation is done using an inverted microscope and the number of dead and live worms is recorded in each session. Hi, I'm Greg Solis. I'm in the Petro Lab in the Department of Chemical Physiology at the Scripps Research Institute.
Today I'll be showing you how to synchronize worms and distribute them in 96 wall microtiter plates. The main advantage of this technique of your existing methods is that allows for the rapid identification of molecules that extend lifespan and C elegance. So let's get started.
To prepare feeding bacteria for C elegance begin on day minus seven by inoculating five milliliters of TB containing 100 micrograms per milliliter and persin, and 0.1 micrograms per milliliter and terin B with a single OP 50 colony incubate overnight at 37 degrees Celsius in a bacterial shaker early the next morning, dilute the overnight culture of OP 51 in 2000 in 300 milliliters of TB containing 50 micrograms per milliliter of ampicillin. Incubate the culture in a bacterial shaker for eight to 12 hours at 37 degrees Celsius until saturation is reached. Do not allow the culture to grow longer than 14 hours.
Transfer the OP 50 into a sterile pre weighted centrifugation tube Pellet the OP 50 by centrifugation for 10 minutes at 3, 500 RPM or 2, 200 times G in a tabletop centrifuge. Discard the S supernatant resuspend the OP 50 pellet and star our water and rep pellet by centrifugation. Repeat this, wash twice after the second.
Wash carefully and thoroughly remove all the remaining water. Weigh the centrifugation tube containing the pellet and subtract the weight of the empty centrifugation tube in order to determine the weight of the pellet. Next, thoroughly resuspend the pellet in S Complete to a concentration of 100 milligrams per milliliter.
No clumps should be left. The concentration of 100 milligrams per milliliter OP 50 should correspond to two times 10 to the 10 bacteria per milliliter. Use the photo spectrometer to determine the number of bacteria per milliliter if the relationship between the optical density and the number of bacteria per milliliter is known.
If necessary, adjust the concentration of the OP 50 feeding solution to two times 10 to the 10 bacteria per milliliter. Finally, store the OP 50 solution at four degrees Celsius until it is used for the worm culture to generate an age synchronous population of worms. Begin late afternoon on day minus six with a five to 10 day old NGM plate on which the worm population consists mainly of starved L one larvae.
Sterilize a metal spatula by shortly heating it over a bunsen burner. Let it cool. Then use the cool spatula to cut out agar chunks from the plate containing the starved worms.
Transfer several of these chunks onto a fresh 10 centimeter NGM plate seeded with OP 50. Incubate the worms for approximately 65 hours at 20 degrees Celsius until the majority of the worm population consists of grave adults. The time it takes for the starved L one animals to grow into grave adults may vary from strain to strain.
After the incubation, collect worms from the 10 centimeter plate by washing them off the plate with five to 10 milliliters of sterile water. Transfer the worm water solution into a 15 milliliter conical tube. Wash the worms by centrifuging for two minutes in a tabletop centrifuge at 1, 200 RPM or 280 times G.Discard the supinate and add 10 milliliters of water.
Repeat the wash step, remove the SUP natant and add five milliliters of freshly prepared bleach. Sodium hydroxide solution incubate for five minutes at RT until the worms break open. Be sure to vortex gently every minute.
Monitor the progress of the reaction under the dissecting microscope as soon as all the adults dissolve. Add five milliliters of M nine buffer to neutralize the reaction. Wash the eggs three times with 10 milliliters of M nine buffer by centrifuging for two minutes at 2, 500 RPM or 1, 100 times G.Then wash the eggs once with 10 milliliters of S complete by centrifuging for two minutes at 2, 500 RPM or 1, 100 times G.Remove the snat.
Add 10 milliliters of S complete and transfer the solution to a fresh 50 milliliter conical tube. Add 30 milliliters of S complete to a final volume of 40 milliliters. Gently shake the tube overnight at room temperature on a mutator or similar device to seed the animal plates the next day around noon.
Under a dissecting scope, check whether the worms hatched during the night. Determine the concentration of worms in the S complete solution by counting the number of worms in 10 microliter drops. Using a dissecting scope count least 10 drops for each sample.Resus.
Suspend the worms at a concentration of 80 to 100 worms per milliliter. In S complete medium, add carbenicillin to a final concentration of 50 micrograms per milliliter and amphotericin B to a final concentration of 0.1 micrograms per milliliter. Shake on a mutator.
If preparing larger quantities of worms, use a 600 milliliter non clon flask with a filter cap At 2:30 PM Add the OP 50 prepared in part one to a final concentration of six milligrams per milliliter. Return the OP 50 to four degrees Celsius. Transfer 120 microliters of the worm OP 50 solution into each well of a 96 well plate with the transparent bottom.
Make sure to keep the worms and suspension while pipetting seal the plate using a tape sealer to avoid contamination and evaporation. Shake the plate on a microtiter plate shaker for two minutes and incubate for two days at 20 degrees Celsius until the animals reach the L four stage. Next to sterilize the animals add 30 microliters of a 0.6 millimolar FUDR stock solution to each well.
This step brings the final volume in each well to 150 microliters and reduces the final concentration of OP 50 from six milligrams per milliliter to five milligrams per milliliter. Seal the plate using tape sealers and shake it for two to three minutes on a microt plate shaker. If the OP 50 was added at two 30 on day minus two, it is important that the FUDR is added before noon return plates to the 20 degrees Celsius incubator by 9:00 AM the next day.
Most of the animals should be grave and contain several eggs. Each add the drugs whose effect on lifespan is to be tested at the desired concentration. If dissolving the drugs in DMSO, the final concentrations of DMSO should not exceed 0.6%as DMSO concentrations higher than 0.6%Shorten the elgan lifespan after addition of the drug.
Seal the plates with tape sealer and shake them for two to three minutes on a microtiter plate. Shaker return the plates to the 20 degrees Celsius incubator. Adding the drugs can occasionally kill a few animals per plate, especially if using a solvent other than water.
Use an inverted microscope to check for dead animals. Generally, there should be less than 10 dead animals per 96 world plate Return plates to the 20 degrees Celsius incubator for two days to allow fresh oxygen to enter the culture. Remove the sealer.
Wait one minute and reseal the plate. Shake the plate for two to three minutes on a microt tighter plate Shaker, repeat once every week on day five of the adult lifespan, add five microliters of the 100 milligram per milliliter OP 50 solution that was prepared earlier to each of the 96 wells to prevent starvation. Worms are scored for lifespan by their movement three times per week from the beginning of the experiment until they die.
Use an inverted microscope with a two x or 2.5 x objective to observe worms in 96. Well plates on day zero at the beginning of the experiment. Count the total number of worms in each well.
Sensor wells that contain more than 18 animals from the analysis as animals in these wells will not have enough OP 50 and will show effects of dietary restriction in each counting session. Record the date and number of animals that move as live animals. Movement and liquid is much easier than on solid media and can be induced by strong lights.
The use of a higher magnification may help to spot very subtle movements like those at the tip of the pharynx. Such subtle movements are often the only movement observed in very old animals. Return the plates to the 20 degrees Celsius incubator.
Repeat the counting sessions every two to three days until all the animals are dead. This is a design of an Excel sheet to record lifespan data for each well. It's coordinate in the plate strain, drug concentration of the drug, and the total number of animals alive on day zero is recorded at the beginning of the experiment.
Record the date as well as the number of living and dead animals three times a week in order to follow the survival of the various populations in each well. This figure shows results of sea elegance, lifespan, curves recorded at 20 degrees Celsius and 25 degrees Celsius. The microtiter based lifespan assay accurately reproduces temperature dependent changes in lifespan.
Similarly, as shown here, the microtiter plate assay reproduces changes in lifespan from mutants reported to have lifespans that differ from wild type N two animals. This figure shows how increasing doses of mirtazapine affect mean lifespan of sea elegance. The data obtained by the microtiter plate assay are quantitative enough to determine dose response relationships Once mastered, this technique can be used to screen large chemical libraries for molecules that extend lifespan and see elegance.
Thanks for watching and good luck with your experiments.
