Pharyngeal Pumping Assay
Materials
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Advanced Preparations
- 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
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Four days after bleaching wash worms off of 10 cm plates with M9 into 1.5 mL Eppendorf tubes.
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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.
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After the final wash, add desired drugs + 10 mM serotonin and wait 20 min.
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Pipette worms and solution onto an unseeded 10 cm plate and wait 1-2 min.
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Pick 5-8 worms without using OP50 to unseeded 6 cm assay plates (GFP+ where needed). Wait 10 min. before counting the pharyngeal pumps.
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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
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One day after bleaching wash worms off of 10cm plates with M9 into 1.5 mL eppendorf tubes.
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Prepare worms by washing worms from 10cm NGM plates into 1.5 mL eppendorf tubes using 1 mL of M9.
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Add desired drugs and wait 20 min.
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Pipette worms and solution onto an unseeded 10cm plate and wait 5 min.
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Pick 5-8 worms to seeded 6 cm assay plates (GFP+ where needed). Wait 10 min. before counting the pharyngeal pumps.
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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.