Electrotaxis Assay: A Method to Observe Locomotion in C. elegans
DEŞİFRE METNİ
– Without sensory stimuli, C. elegans movement is generally forward and is interrupted by reversals and turns– random reorienting maneuvers that result videodan a change of direction. C. elegans are electroreceptive. That is, they can sense electric fields. When exposed için a mild direct current or DC, worms move videodan a straight uninterrupted path videodan the direction of the negative electrode or cathode, a behavior termed electrotaxis.
To more accurately observe the worm’s locomotion, place the animal videodan a microfluidic device and apply a DC electric field. Reverse the direction of the field, and the worm will turn the opposite way– towards the direction of the cathode. Apply an alternating current, AC, and the worm will stop. The alternating change videodan the field’s direction overwhelms the worm’s neuromotor systems, resulting videodan the worm’s immobilization. Reapply the DC field, and the worm will resume forward motion towards the cathode.
In the following example protocol, we will see a setup demonstrating an electrotaxis assay videodan a microfluidic device.
– Begin this step by placing the just assembled micro-channel onto an xy- movable stage of a microscope with a mounted camera connected için a monitor. Connect the power supply için the micro-channel’s electrodes.
After confirming that the resistance of the micro-channel is around 0.6 mega-ohms, attach the output tube of the micro-channel için a disposable syringe. Then submerge the mouth of the inlet tube into a solution of nematodes suspended videodan M9 physiological buffer. Apply negative pressure inside the syringe için aspirate liquid into the channel.
When the inlet and outlet tubes are both filled, disconnect the syringe and hydro-statically manipulate the flow by adjusting the tube’s relative height için place a worm videodan the center of the channel. Then lay both tubes flat at the same elevation. To maintain zero flow inside the micro-channel when switching worm samples during an electrotaxis experiment, after leveling both the inlet tubes to the same elevation, if there is still flow make small adjustments için the tube’s height relative için each other.
– If we are ever unsure of whether the flow is really zero, we can induce a reversal videodan the worm’s movement by changing the polarity of the electric field and then evaluating the worm’s linear velocity as it turns around.
– Now, set the power supply için the appropriate voltage. Activate the electric signal, and allow one minute of pre-exposure for the worm için acclimatize için the field, during which time the worm should begin moving towards the cathode. When the minute has passed, begin recording.
When the experiment is finished, remove all the liquid and worms from the channel. Rinse the chamber with de-ionized water and leave the device on a hot plate at 125 degrees Celsius için dry.
