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Levamisole is a synthetic anthelmintic drug extensively employed in veterinary medicine to treat parasitic infections caused by nematodes. As an anthelmintic, it targets the nematode acetylcholine ion-channel receptors (Martin et al). Besides its medical applications, Levamisole also serves as a widely used reagent in various research fields, particularly in C. elegans paralysis assays. Its ability to induce paralysis in these nematodes makes it invaluable for studying neuronal function and muscle physiology. To provide insight into toxicological experimentation, we present an example below utilizing this compound. Researchers can use this example as a framework to conduct their own toxicity studies using different soluble drugs.

Materials

• Young adult worms
• 96 well “U”-bottom plate with lid.
• M9 buffer.
• OP50.
• Levamisole – Stock solution: 100mM in ddH20.
• Synchronized populations of young adult worms.
• wMicrotracker ONE.

Protocol Brief:

PROTOCOL

1. Grow synchronized populations of young adult worms in seeding NGM plates (OP50).
2. Collect worms from plates using M9 buffer and transfer them in a sterile 15 ml tube.
3. Let the worms settle. Remove the supernatant taking care not to disturb the pellet.
4. Perform a wash with 5 ml of M9 buffer. Briefly shake or invert the tube. Repeat step 3.
5. Add 3 ml of M9 buffer.
6. Count the number of worms in a volume of 10 µl and adjust the volume to obtain a concentration of [20worms/90µl]. Adjust volume with M9 and add OP50 to 1 mg/ml final concentration.
7. Transfer per well 90μl of the worm solution to a 96 well microplate using multichannel pipette.
8. Add 10µl of a 10X concentrated solution of chemicals to test and gently shake microplate by hand. Include a control without compounds.
9. Register worm activity using WMicrotracker ONE.
10. Generate the data report using WMicrotracker One software and plot using MSExcel.

Expected Results

NOTES

• Perform at least three technical replicates and at least two biological replicates.
• Before activity measure, stimulate the 96 well plate with worms for 5 second by gently shaking by hand.
• A basal record can be made before carrying out the treatment. This value can be used to relativize the data after treatment.
• If dissolving the drugs in DMSO the final concentrations of DMSO should not exceed 1%.