$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
This protocol is an excerpt from Tong et al, Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion, J. Vis. Exp. (2013).
1. Electrotaxis Experiment
- Place the microchannel on the stage (preferably XY-movable) of a microscope with a mounted camera connected to a monitor (Figure 1).
- Connect the power supply or amplifier's output wires to the microchannel's electrodes. A simple DC power supply is sufficient if only a DC signal is desired, but an amplifier connected to a function generator allows application of pulsed DC and AC signals as well.
- Attach the microchannel's output tube to a disposable syringe. Submerge the mouth of the inlet tube in M9 physiological buffer and gently aspire liquid into the channel by applying a negative pressure inside the syringe (either manually or using a syringe pump). When the inlet and outlet tubes are both filled with M9, disconnect the syringe from the tube. Level both tubes to the same height to prevent hydrostatically driven flow.
- Apply a DC voltage to the channel and ensure that resistance (R= V/I) is around 0.6 MΩ (for a 50 mm long, 0.3 mm wide and ~0.1 mm deep microchannel).
- If satisfied with the channel's integrity, follow the above steps to load worms from a diluted suspension into the channel.
- Disconnect the syringe and hydrostatically manipulate the flow by adjusting the tubes' relative height. Use this method to place a worm in the center of the channel and then lay both tubes flat at the same elevation.
- Set the power supply to the appropriate voltage: 4-12 V/cm for L3 stage animals, 4-10 V/cm for L4s, and 2-4 V/cm for young adults. Activate the electric signal and allow 1 min of pre-exposure for the worm to acclimatize to the field. The worm should begin moving towards the cathode. When the minute has passed, use the camera to begin recording.
- For AC and pulsed DC experiments, the maximum responsive electric field can be adopted from above and frequency and duty cycle of the signal can be modulated as desired.
- When experiment is finished, remove all liquid (and worms) from the channel, rinse it with dH20, and leave the device on a hot plate at 125 °C to dry.
- Extract locomotory data from recorded videos manually using NIH ImageJ (http://rsbweb.nih.gov/ij/) or custom MATLAB-based worm tracking software.
