An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms

1. PolyQ-mediated neurotoxicity assay

1. Treatment of C. elegans with test samples
   1. To prepare nematodes for the polyQ neurotoxicity assay, transfer 300-500 synchronized L1 larvae of HA759 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 three replicate wells for each treatment.
   2. Seal the plate with parafilm and incubate at 15 °C and 120 rpm for 3 days.
   3. Collect the nematodes by centrifugation and wash 3-5 times with M9 buffer. Resuspend the nematodes in the M9 buffer for use in neuronal survival and avoidance assays.
2. Preparation of agarose pad
   1. Add 2 g of agarose to 100 mL of M9 buffer (2%, w/v) and heat the agarose solution in a microwave to near-boiling.
   2. Dispense 0.5 mL of melted agarose onto the center of a 1 mm thick microscopy glass slide placed between two pieces of 2 mm thick glass plates. Cover with another slide vertically. Once the agarose cools down and is solidified, gently remove the top slide.
3. ASH neuronal survival assay
   1. Add a drop of 20 mM sodium azide onto the agarose pad. Transfer 15-20 HA759 nematodes into the drop to immobilize them.
   2. Place a coverslip gently over the nematodes. Keep the slide under a fluorescence microscope fitted with a digital camera. Select a 40x objective lens and FITC filter to detect GFP-positive ASH neurons in the head region of the nematodes.
   3. Select more than 50 nematodes in each group randomly to count the number of nematodes with GFP-labeled bilateral ASH neurons in their head region. Calculate the survival rate of ASH neurons:
      Neuronal survival (%) = 
      Where N_survival and N_total are the number of nematodes with GFP-positive ASH neurons and the total number of tested nematodes in each group, respectively.