An Assay to Study Effect of Test Compound against PolyQ-Mediated Neurotoxicity in Worms
Transcript
To prepare nematodes for the polyQ neurotoxicity assay, transfer 300 to 500 synchronized L1 larvae of HA759 nematodes to each well of a 48-well plate with 500 microliters of S medium containing OP50 strain of E. coli and 5 milligrams per milliliter of astragalan. After sealing the plates, incubate, harvest, and resuspend the nematodes in M9 buffer, as demonstrated.
Now prepare an agarose pad by adding 2 grams of agarose to 100 milliliters of M9 buffer, and heat the solution in a microwave to near boiling. Dispense 0.5 milliliters of melted agarose onto the center of a 1-millimeter thick microscopy glass slide, placed between two pieces of 2-millimeter thick glass plates, covering with another slide vertically. Gently remove the top slide once agarose cools down and is solidified.
Begin the ASH neuronal survival assay by adding a drop of 20 millimolar sodium azide onto the agarose pad, and then transferring 15 to 20 HA759 nematodes into the drop to immobilize them. Place a cover slip gently over the nematodes.
Now, 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.
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, and then, calculate the survival rate of ASH neurons.
