The overall goal of this procedure is to examine antipsychotic drug effects on the development and pharyngeal pumping of sea elgan. This is accomplished by first growing a population of adult nematodes and from them generating a synchronized population of animals. The next step is to transfer synchronized eggs or adults to drug laced assay plates.
The final step is to observe and score the effects of the drugs on the worms. Ultimately, the results can show that antipsychotic drugs delay development and inhibit pharyngeal pumping in drug exposed animals. Though this method can provide insight into the mechanisms of action of antipsychotic drugs, it can also be applied to study other small molecules and even large molecules such as lipids Have ready a drug of interest with a known working concentration.
Begin by loading a 12 well plate with two milliliters of NGM, medium per well. Set the plate aside to harden at room temperature. Next, pick a colony of OP 50 bacteria and transfer it to a 50 milliliter bottle of LB culture.
The bacteria overnight at 37 degrees Celsius with shaking at 220 RPM the next day into each well of the prepared plate. At 20 microliters of the bacterial culture, set the plate to incubate overnight at 37 degrees Celsius the following day. Load each seated well with 80 microliters of drug of interest at the working concentration.
In this case, the drug is C Clozapine. Be sure to vortex the solution before taking aliquots. Swirl the solution to distribute it evenly in the well and allow it to absorb at room temperature with the plate lid off.
Then proceed with the assay or store the plate at 20 degrees Celsius overnight and start the assay the next day. Begin by collecting eggs from the genotype of interest. Start with a population of worms with many GR adults.
Wash the worms off in a 3.5 centimeter plate using M nine buffer. Then spin them down in a 15 milliliter tube at 2000 RPM for one minute. Remove the supernatant via aspiration and replace it with five milliliters of bleach.
Then gently disrupt the adults by inverting the tube and wait for the animals to start dissolving continuously. Observe the dissolving worms under the scope. When about half the worms have dissolved.
After about five minutes, spin down the tube to collect the eggs, aspirate the supernatant, and quickly replace it with M nine buffer. Then spin the tube down again as before and remove all but about 100 microliters of the M nine supernatant. To suspend the eggs, vortex the tube.
Then transfer two microliters of the suspension to an NGM plate and count the number of transferred eggs. Then load the drug treated plate with 30 to 35 eggs per well and incubate the plate at 20 degrees Celsius for 24 hours. After 24 hours, score the number of hatched eggs.
If more than 25 worms are present in any, well remove the excess worms. Return the worms on the plate to the incubator for later observation at 24 hour intervals each day, score the developmental stage of the worms in the wells based on the size of the worms and the shape of their vulvas. Robust effects are usually seen on the third and fourth days.
The experiment should last about a week. For this protocol, have prepared drug test plates as previously described the day before, initiating the assay. Pick 50 L four stage animals for each genotype in the experiment.
Transfer them to NGM plates and incubate the plates for 24 hours at 20 degrees Celsius. The next day, every 15 to 20 minutes, load a test well with 10 animals after a group has been exposed to the drug for half an hour. Start observing their pharyngeal pumping under a dissection microscope.
First, find a worm, then count its pharyngeal pumps for 20 seconds. Once scored, pick the worm off the plate. Score all the worms in the well in the same manner.
The expected results of conducting the experimental delay. Lethality assay with wild type controls should show that unlike the typical growth rate to the grave, adult stage, animals exposed to c clozapine are still in the young larval stages or are dead testing mutants on different clozapine concentrations revealed suppressor mutants such as mute one and enhancer mutants such as MU two. Universally, the lethality of mute one was lower than wild type, and the lethality of MU two was higher as expected compared to the control surviving mute one animals are more able to develop, whereas mute two survivors are more delayed.
Further examination revealed that Clozapine exposure reduced the pharyngeal pumping rates in a concentration dependent manner. However, the decreased rate of mute one was less than that of wild type, while the decreased rate of mute two was greater than that of wild type. After watching this video, you should have a good understanding of how to perform antipsychotic drug assay on a small scale.
The message can also be scaled up to conduct a large scale genetic screen.
