To begin, remove the culture plate containing adult hermaphrodite female worms from the incubator. After synchronizing him-5 worms, wash the worms five times with M9 buffer. Transfer the one-day-old adult males from the seeded plate to a tube containing M9 buffer.
Place the worms on an unseeded NGM plate to eliminate residual bacteria and prevent interference from food during the assay. Prepare a chemoattraction assay plate with 1.5%auger, 25 millimolar sodium chloride, 1.5 millimolar Tris base, and 3.5 millimolar Tris hydrochloride. Heat the auger in the chemoattraction solution in a microwave until completely dissolved.
After cooling, use a pipette to evenly distribute the chemoattraction auger solution into petri dishes. Leave the lids open in a clean area to allow the surface of the auger to dry slightly. Once the surface has dried appropriately, close the lids.
To conduct the chemoattraction assay, mark three distinct spots on the lid and the underside of the petri dish. Apply two microliters of one molar sodium azide to each experimental and control spot on the plate. Pick 20 healthy and freely moving male worms with a worm picker.
Under a dissecting microscope, release the worm at the starting point. Quickly add two microliters of M9 buffer to the control spot and two microliters of sex pheromone to the experimental spot on the lid. Gently close the lid and place the plate in a quiet, temperature-stable area.
After 30 minutes, score the assay by counting the number of worms at each spot. For an enhanced assessment of chemotaxis, record the worms'trajectories using a camera. The chemotaxis index for him-5 males was consistently above 0.5, indicating a strong attraction to the sex pheromone source, while the chemotaxis index for SRD-1 males was around zero.
Successful chemoattraction assays showed him-5 males moving closer to the pheromone source over time, with color-coded trajectories indicating time, distance, speed, straightness, and direction correctness.
