PolyQ Aggregation Assay to Identify Neuroprotective Effect of a Compound in Worms
Transcript
Begin by transferring 300 to 500 synchronized L1 larvae of AM141 nematodes to each well of a 48-well plate with 500 microliters of S medium containing OP50 strain of E. coli, and five milligrams per milliliter of astragalan. Seal the plate with parafilm, and incubate it at 20 degrees Celsius and 120 rotations per minute for desired time intervals.
Transfer the nematodes in a sterile 1.5-milliliter microcentrifuge tube, and wash with M9 buffer three times by centrifugation, to remove the remaining OP50 cells. Then, resuspend the AM141 nematodes in M9 buffer.
Now, transfer 10 to 15 nematodes into each well in a 384-well plate, setting 10 replicate wells for each treatment, and add 10 microliters of 200 millimolar sodium azide to each well, to paralyze the nematodes by allowing them to settle down to the bottom.
Place the plate in a high-content imaging system to acquire fluorescent images. Open the image acquisition software, and set up the parameters mentioned in the text manuscript. Analyze the image data by opening the "Review Plate Data" window and selecting the "Test Plate" for image analysis.
Double-click on a test well to display its image. Then, select the "Count Nuclei" as the analysis method, and click on the "Configure Settings" button. Define the source image from the FITC channel and select the "Standard Algorithm."
Set the image analysis parameters as described in the text manuscript, and test them to optimize the method of analysis. Save the settings and run the analysis on all the wells. Export the "Total Nuclei" as the total number of Q40::YFP aggregates in each well.
Count the number of nematodes in each well, and calculate the average number of Q40::YFP aggregates per nematode in each group. Then, calculate the inhibition index.
