PolyQ Aggregation Assay to Identify Neuroprotective Effect of a Compound in Worms

1. PolyQ aggregation assay

1. Preparation of nematodes for the polyQ aggregation assay
   1. Transfer 300-500 synchronized L1 larvae of AM141 to each well of a 48-well plate with 500 µL of S medium containing OP50 (OD₅₇₀ of 0.7-0.8) and 5 mg/mL of astragalan, typically one well per treatment for one time point.
   2. Seal the plate with parafilm and incubate at 20 °C and 120 rpm for 24, 48, 72, and 96 h.
   3. Harvest the nematodes in a sterile 1.5 mL microcentrifuge tube and wash with M9 buffer >3 times by centrifugation (1000 × g for 1 min) to remove the remaining OP50. Resuspend the AM141 nematodes in M9 buffer, and keep them ready for image acquisition.
2. Acquisition of fluorescent images and data analysis
   NOTE: Data are automatically analyzed with this high-content imaging system. If an automated imaging device is not available, a conventional method to prepare an agarose pad for image acquisition can be used to achieve similar performance by using a common fluorescent microscope.
   1. Transfer 10-15 nematodes into each well in a 384-well plate (final volume 80 µL per well). Set 10 replicate wells for each treatment.
   2. Add 10 µL of 200 mM sodium azide to each well to paralyze the nematodes and allow them to settle down to the bottom (5-10 min).
   3. Place the plate in a high-content imaging system to acquire fluorescent images (see the Table of Materials for device and software information).
   4. Open the image acquisition software and set up the following parameters.
      1. Open the Plate Acquisition Setup window and create a new experiment set and name.
      2. Select Magnification as 2x, Camera binning as 1, and Plate type as 384-well plate.
      3. Set the wells and sites (single site) to be visited.
      4. Select the fluorescein isothiocyanate (FITC) filter and enable image-based focusing options.
      5. Set the Exposure as 300 ms.
         NOTE: The above settings can be tested and adjusted to optimize the imaging parameters.
      6. Save Image acquisition settings and click on the Acquire Plate button to run.
   5. Analyze the image data by using the image analysis software.
      1. Open the Review Plate Data window and select the Test plate for image analysis.
      2. Double-click on a test well to display its image.
      3. Select the Count Nuclei as the analysis method and click on the Configure Settings button to bring out a window for the following settings.
      4. Define the source image from the FITC channel and select the Standard Algorithm.
      5. Set the image analysis parameters as follows: approximate minimum width = 10 µm (= 2 pixels); approximate maximum width = 50 µm (= 12 pixels); intensity above local background = 1,000-2,000 graylevels.
      6. Test the current settings to optimize the method of analysis.
      7. Save the settings and run the analysis on all the wells.
         NOTE: It takes ~20 min to finish analysis for one 384-well plate.
      8. Export the Total Nuclei as the total number of Q40::YFP aggregates in each well.
   6. Count the number of nematodes in each well.
   7. Calculate the average number of Q40::YFP aggregates per nematode in each group, and apply a nonlinear curve fit to the data from each time point.
   8. Calculate the inhibition index:
      Inhibition index = (N_control – N_sample) / Ncontrol
      Where N_control and N_sample are the average number of Q40::YFP aggregates in the control and the treatment groups, respectively.