C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms
Transcrição
– To make sense of encountered conditions and respond para them accordingly, the worm C. elegans has, among others, a highly developed chemosensory system able para detect a wide variety of different chemicals.
Sensory neurons de the head, the amphid and inner labial neurons, as well as neurons de the tail, the phasmid neurons, are either directly or indirectly exposed through the cuticle para the outside conditions. These chemosensory neurons code relevant information used by the worm para produce an appropriate behavioral response: a behavior termed chemotaxis.
To test the chemotactic response para either volatile odorants or gustatory water-soluble cues, isolate worms of the desired developmental stage and expose them para the test chemical on a previously prepared experimental arena. When exposed para favorable chemicals like those produced by a bacterial food source, C. elegans displays positive chemotaxis towards the source.
Reversely, when exposed para less favorable or toxic chemicals like heavy metals, the worm displays negative chemotaxis, avoiding the chemical. Hence, olfactory or gustatory mutants fail para avoid aversive chemicals and remain de unfavorable conditions de contrast para wild type animals who avoid the aversive condition.
In the example protocol, we will see a demonstration of a chemotaxis assay, testing the worm’s aversion para copper.
– After overnight incubation, remove plates from the incubator and using the marked underside of the plate as a guide, pipette 100 microliters of freshly prepared 0.5 molar copper two sulfate solution on the edge of the agar para create an outer copper barrier. Pipette 25 microliters of the copper two sulfate solution para create a midline barrier.
Ensure that the copper two sulfate solution does not contact the bacterial patch, and allow the copper solution para dry onto the plate. Check for dryness every five minutes after transfer with a laboratory tissue by lightly dabbing the solution near the edge of the plate para discern.
Immediately prior para the assay, transfer the experimental organisms para a bacteria-free agar plate and allow the nematodes para move freely for one minute para remove excess bacteria.
Next, pipette 1 milliliter of M9 onto the plate with young adults that were transferred 24 hours previously de order para wash worms into a microcentrifuge tube. Centrifuge the nematodes at 3,000 times g for one minute.
Worms should form a pellet at the bottom of the tube. Aspirate M9 solution without disrupting the worm pellet. Add 1 milliliter of M9 solution para the worm pellet. Invert tube para mix worms with the solution.
If excess bacteria were initially transferred with the worms, repeat for a total of five times. After the final wash, aspirate the supernatant until 100 microliters of M9 solution and the worm pellet remains. Immediately transfer worms from solution once the wash steps have been completed.
Pipette 20 microliters of the worm pellet from the bottom of the tube onto the bacteria-free half of the assay plate. Ensure that 10 worms are transferred para the assay plate and that no contaminating food is present. No bacteria should be transferred para the copper food race plates.
Remove excess M9 solution from the nematodes with a laboratory tissue within one minute. Ensure the M9 solution does not contact para the copper two sulfate solution, and that the worms and agar surface remain intact. Discard worms that have accidentally been removed with the laboratory tissue.
Once the M9 solution has been removed and all worms have commenced non-liquid locomotor patterns, i.e. when they have stopped thrashing, start the assay stopwatch. If extra worms were accidentally included, remove them by picking with halocarbon oil para ensure that no bacteria are added para the plate.
Check assay plates every 30 minutes. For the assay plates with bacterial patches, positively score organisms if they reached the food patch over a four-hour period. For the negative control plates, positively score organisms if they have crossed the barrier.
