The overall goal of this technique is to test the effects of different compounds on the model organisms C.elegans in a high throughput assay. This method can help answer key questions in the chemical, biology, and aging field, such as do specific chemical structures extend the lifespan of C.elegans? The main advantage of this technique is that it allows for the screening of numerous compounds in a time efficient and rapid manner.
To prepare mass culture plates for the production of large numbers of worms, in a laminar flow cabinet, first dispense 30 milliliters of 60 degrees Celsius nematode growth medium agar onto 10 centimeter culture plates for overnight solidification. The next morning, add two milliliters of concentrated E.coli onto each plate, tilting and rotating the plate to spread the bacteria completely across the plate surface. When the mass culture plates have dried, store them at four degrees Celsius for up to two weeks.
To prepare high throughput assay culture plates for screening, use an automated eight channel dispenser to add 0.15 milliliters of molten nematode growth medium agar into each well of a 96 well plate. Allow the agar to solidify overnight. Then store the high throughput assay plates at four degrees Celsius for up to two weeks.
To set up the C.elegans cultures, use a worm pick and a dissecting microscope to transfer 20 eggs onto each mass culture plate and incubate the plates at 20 degrees Celsius for six days. On the seventh day, use a dissecting microscope to visually confirm the presence of eggs in the uterus. To collect the gravid adults, use a glass pipet to add two to three milliliters of S Basal solution per plate and swirl the solutions around each plate by hand.
Use the glass pipet to transfer the worms into a 15 milliliter tube and pellet the worms by centrifugation. Re-suspend the worms in 10 milliliters of hypochlorite solution and rapidly shake the tubes up and down for five seconds. Then, incubate the worms for five minutes at room temperature with shaking every 2.5 minutes.
At the end of the incubation, centrifuge the worms again. The pellets should be yellowish brown. Replace the supernatants with 10 milliliters of fresh hypochlorite solution and vigorously shake the tube again.
Incubate the worms for another one to three minutes with occasional shaking. When only eggs remain, centrifuge the tubes and aspirate the supernatants. The pellets should be white with no brown coloration.
Using sterile technique, open the tubes in a steril laminar flow cabinet and add 14 milliliters of S Basal solution to each pellet. Wash the pellet three times with 14 milliliters of S Basal. After three washes by centrifugation, re-suspend the eggs in two to three milliliters of fresh S Basal solution.
To prepare L1 stage larval C.elegans, transfer the supernatants into one sterile glass Petri dish per tube and use two to three milliliters of S Basal solution to rinse the remaining eggs from the tubes into each dish. Add five milliliters of S Basal solution to each dish to ease crowding and place the dish on an orbital shaker at 20 degrees Celsius and 20 rpm for 16 to 24 hours to encourage hatching. All of the hatchlings will arrest at the first larval stage due to a lack of food.
Using a serologic pipet, transfer the L1 larvae into a 15 milliliter tube for their centrifugation. After all hatched L1 have been consolidated through repeated centrifugation and aspiration, re-suspend larva pellet with 10 milliliters of sterile S Basal solution. Then, transfer 10 microliters to an unseeded plate and count the number of larvae that are present on the plate.
Repeat this 10 times to obtain the average density of L1s in your solution. With a sterile serological pipet, determine the total volume of solution containing the L1s. Next, use the volume of the L1 solution and the density of L1s in the solution to calculate the total number of L1s present in your solution.
To plate the worms in a 96 well high throughput assay plate, dilute the L1 suspension to one L1 larva per microliter in a sterile, round media bottle, equipped with a moderately slow turning stir bar and allow the assay plates to warm to room temperature. Next, in a laminar flow cabinet, add five microliters of concentrated E.coli to each well, followed by 10 microliters of L1 suspension per well to yield an average of 10 worms per well. When all of the worms have been plated, cover the plates and incubate worms for two days at 25 degrees Celsius, to allow the larvae to develop into adults.
At the end of the incubation, bring the chemical library plates up to room temperature from freezing. Gently shake the thawed plates at 60 rpm on a microplate shaker for one minute immediately prior to use. Using an automated 96 well liquid handler, transfer 0.75 microliters of compound from the library plate to the assay plate.
When all of the wells have been treated, air dry the compound from the surface of the plates in a laminar flow cabinet for four to eight hours, removing the individual plates as soon as they are dry. Seal the dried plates with transparent film and lid them. Then spin the plates for 45 seconds at 1000 G and store them in the inverted position at 25 degrees Celsius.
To test C.elegans'responses to the compounds of interest, a negative control plate can be used to score the total survival until 95%of the control animals are dead, at which point all of the test plates can be scored. For example, in this study, 57 hits were observed from the retest library of 179 compounds, demonstrating that 32%of the retest chemicals tested positive in total. Binning of the averaged percentage alive scores for the negative control and test wells indicated that the test wells outperformed the control wells.
Small-scale screens can also be performed, as demonstrated in this representative test assay in which 50 micromolar and 100 micromolar concentrations of the test compound were used. Re-scoring these small-screen assay test plates when greater than 99%of the negative control treated animals were dead, indicated that the positive control wells far outperformed the negative control and test wells in that the test wells performed slightly better than the negative controls. In this experiment, the latter result was biased by the indication that many of the test wells appeared to exhibit toxicity relative to the control treatment, thereby confounding this simple interpretation.
Once mastered, this technique can be completed in two to four weeks if performed properly. When performing this procedure, it is important to maintain the integrity of the assay plates, making sure they're pristine before they're used. Following this technique, additional questions can be answered, such as, which of these compounds induces mytophagy?
For a mytophagy assay, a mytophagy reporter strand would be used. After watching this video, you should have a good idea of how to perform a high throughput chemical screen using C.elegans as a model organism.
