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Pharyngeal Pumping Assay

Materials

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Advanced Preparations

  1. Bleach synchronize worm strains and hatch 1,000 embryos on unseeded 10cm NGM plates for L1 assays or seeded 10 cm NGM plates for adult assays.

Adult Assay Protocol

  1. Four days after bleaching wash worms off of 10 cm plates with M9 into 1.5 mL Eppendorf tubes.

  2. Wash and prepare worms:

    a. Wash worms from 10 cm NGM plates into 1.5 mL eppendorf tubes using 1 mL of M9.

    b. Allow worms to settle for 2 min.

    c. Remove supernatant without disturbing the worm pellet.

    d. Add 1 mL of M9 and invert to wash worms.

    e. Repeat steps b-d two more times, for a total of three M9 washes.

  3. After the final wash, add desired drugs + 10 mM serotonin and wait 20 min.

  4. Pipette worms and solution onto an unseeded 10 cm plate and wait 1-2 min.

  5. Pick 5-8 worms without using OP50 to unseeded 6 cm assay plates (GFP+ where needed). Wait 10 min. before counting the pharyngeal pumps.

  6. Use the Axio injection scope to count pumps for 10 sec. One pump is the movement of the terminal bulb down and back to its starting position.

L1 Assay Protocol

  1. One day after bleaching wash worms off of 10cm plates with M9 into 1.5 mL eppendorf tubes.

  2. Prepare worms by washing worms from 10cm NGM plates into 1.5 mL eppendorf tubes using 1 mL of M9.

  3. Add desired drugs and wait 20 min.

  4. Pipette worms and solution onto an unseeded 10cm plate and wait 5 min.

  5. Pick 5-8 worms to seeded 6 cm assay plates (GFP+ where needed). Wait 10 min. before counting the pharyngeal pumps.

  6. Use the Axio injection scope count pumps for 10 sec. One pump is the movement of the terminal bulb down and back to its starting position.