So this method can help to answer key questions in a drug discovery process about how a specific drug behaves in a living organism. The main advantage of this technique is it allows large sample sizes to be screened quickly and efficiently. This technique is already having a major impact in drug discovery by enabling a highly quanititative assessment of the effects of drug candidates.
This method provides novel mechanistic insights into the onset and progression of misfolding diseases. Furthermore, this method can also be applied to other systems, such as aging and genetic screenings. We're very excited by this opportunity to introduce new physical methods into the space of drug discovery.
These demonstartions of this method is critical as the screening steps require high familiarity with this novo automatic technique. Demonstrating the procedure will be Sam Casford, a search assistant from our laboratory. Before beginning the procedure, add 2.2 millileters of each drug compound at the appropriate concentration to six 5-fluorodeoxyuridine or FUDR plates per worm strain, by carefully spreading the compound over the whole surface of the plate, and dry the plates under sterile conditions.
While the plates are drying, use 15 milliliters of M9 buffer to wash the worms from five rich nematode growth factor plates, and transfer the liberated worms into a conical tube for centrifugation. Re-suspend the nematodes in three milliliters of fresh M9 buffer for quantification of the number of worms in the L4 larval stage, and seed 700 L4 larvae onto each of the six dry FUDR plates per worm strain, per compound. When all of the nematodes have been seeded, place the plates containing the strains for which parlysis is induced by raising the temperature at 24 degrees celsius for 24 hours.
The next day, turn on the stage lights of the parallel worm tracker, and clean the glass stage of the tracker with 70 percent ethanol. If no visible residue remains, remove the lens cap and use an air duster to clean the imaging lens. Confirm that the camera is correctly plugged in and installed, and start recording with the image capture software.
Adjust the camera settings to record 20 frames per second, with the appropriate mono-16 recording parameters. Use an empty, nine centimeter petri dish to ensure that the stage is set to the correct height, and adjust the stage if necessary. Under sterile conditions, add three milliliters of M9 solution to a motility screening plate, and use two milliliters of M9 solution to wash one third of the surface area of two nematode loaded FUDR plates from the same experimental group into the screening plate.
Then, place the screening plate onto the tracker stage. Use a single worm to focus the camera, and begin recording the animals. When the recording is complete, discard the motility plate, and mark the FUDR plates as being used one time before returning them to the incubator.
To analyze the video data, open the parallel worm tracker data analysis software graphic user interface, and use the browsing function to load the video of interest. Select an output destination folder, and confirm that 600 to 1200 frames are set for a 30 second analysis. Insert a 0.029 pixel to millimeter conversion factor for the imaging of a nine centimeter petri dish at full resolution, and select the keep dead tracking algorithhm.
Under the locating parameters tab, set the Z-use images to 100, the Z-padding to 3, the standard pixels to 54, the threshold to 9, the opening to 1, and the closing to 2. Adjust the filtering parameters to a minimum size of 20, a maximum size of 180, and a worm like of 0.94. Set the forming trajectories parameters to a maximum distance move of 10, a minimum length of 150, and a memory of 10.
Set the bends and velocity parameters to a bend threshold threshold of 1.8, a minimum bends of 0, and a frames to estimate velocity of 150. Tune the dead worm statistics to a maxmim beat per minute of 5, and a maximum velocity of 1. Select the output folder, and select the number of output frames to 50, and the font size to 10.
Use the example function to test the parameters and output the sample images to check whether the worms are visible throughout the eight thresholding steps. Then initiate the Start Job"function, and combine the individual results for the whole data set to analyze the test result files, saving the results via the export to TSV function. This method allows the characterization of nematode fenotypes for large population studies of various worm models of neurodegenerative disease, such as Frontotemporal Dementia, Parkinson's Disease, Alzheimer's Disease, and Amyotrophic Lateral Sclerosis.
As well as the characterization of the effects of potential therapeutic molecules, using worm models of Parkinson's and Alzheimer's Disease. The high accuracy of these measurements is achieved by increasing the number of worms that can be analyzed compared to traditional methods. While performing this procedure, it's important to remember to minimize the time between the addition of the worms to the motility and the beginning of the tracking.
Following this procedure, other methods can be performed to supplement the results and to link with protein aggregation. After it's development, this technique paved the way for researchers in the field of neurodegenerative and misfolding diseases, such as Parkinson's and Alzheimer's, to explore the right discovery applications. Don't forget that working with FUDR can be extremely hazardous and the precautions, such as wearing nitrile gloves, should always be taken while performing this procedure.
