The overall goal of this procedure is to longitudinally image dynamic processes in adult nematodes under controlled environmental conditions for the tracking of worm behavioral responses and the kinetics of worm gene expression. This method can help answer key questions about how animals adapt to changes in their environment, including differences in core behaviors, gene expression, memory and learning. This technique allows the multiplex longitudinal observation of individual animals under dynamically controlled environments, as well as the simultaneous sampling of multiple aspects of adaptation at a high resolution.
The implication of this technique extends towards a quantitative understanding of animal adaptation, as acquiring high precision kinetic data can facilitate mathematical modeling and the formulation and testing of complex hypotheses. To set up the device for the procedure, first connect the outlet syringe tip to a 50 centimeter piece of syringe tube. To make an adapter needle, use pliers to pull the needle shaft from the needle hub of a 20 gauge by one half inch needle and use a Bunsen burner to remove the remaining plastic residue.
Holding both ends of the needle with pliers, bend the adapter needle according to the geometry of the experimental setup and plug the needle halfway into the other end of the outlet syringe tube. Next, connect one buffer inlet syringe tip directly to a three-way valve and connect a buffer inlet syringe to a one meter piece of syringe tube. Inserting the adapter needle halfway into the open end of the tube, connect the buffer exchange syringe to a 50 centimeter piece of syringe tube via the syringe tip.
Connect the syringe tube to the remaining port of the three-way valve and close the valve. Using an adapter needle, connect the worm inlet syringe to a one meter piece of syringe tube as just demonstrated. Then, rinse all of the materials two times with S-medium and fill the rinsed syringes and tubes with fresh S-medium, taking care to remove any air bubbles.
Now, connect the outlet syringe tube to the device outlet and plug the adapter needle at the end of the outlet syringe tube to the outlet port. Inject a small volume of S-medium through the outlet to fill the device until S-medium comes out of both the buffer and worm inlets. Connect the buffer inlet syringe tube to the buffer inlet port of the device and plug the worm inlet with a stainless steel dowel pin with a one thirty second of an inch diameter.
Mount the microfluidic device on an inverted fluorescence microscope with a motorized stage and tape a syringe pump to a shaker near the microscope so that the pump does not jiggle while shaking. Then, load the buffer inlet syringe onto the syringe pump and set the pump to deliver a continuous flow of S-medium into the device at three microliters per minute flow rate. To load the bacteria into the microfluidic device, first load a new syringe with a high density suspension of E.coli OP50.
Turn the syringe vertically, and tap the barrel several times to collect all of the air bubbles at the top of the syringe. Turn off the orbital shaker and close the three-way valve of the buffer inlet syringes. Replace the buffer inlet syringe with the bacteria-loaded syringe and push out all of the air collected at the top of the syringe until a little bit of the bacterial suspension enters the buffer exchange system and no bubbles remain in the OP50-loaded syringe or the valve.
With the valve closed, connect the bacteria-loaded syringe to the syringe pump and turn on the orbital shaker. Set the shaking speed to approximately 200 RPM and the flow rate to 100 microliters per minute. Once the OP50 buffer has spread throughout the device, set the flow rate back to three microliters per minute.
To load the device with nematodes, first fill a 650 microliter low-binding micro-centrifuge tube with 100 microliters of fresh OP50 suspension, and use a worm pick to individually transfer 20 to 30 age-synchronized young adult worms from a nematode growth medium agar plate to the tube. Fill the worm inlet syringe and syringe tube with fresh OP50 suspension and inject approximately 500 microliters of suspension into the micro-centrifuge tube. Then, draw the worm suspension into the worm inlet syringe tube and connect the worm inlet syringe to the worm inlet.
Inject all of the worms through the worm inlet syringe tube into the microfluidic device and disconnect the worm inlet syringe and syringe tube. Plug the worm inlet with a pin and disengage the buffer inlet syringe from the mechanical pump. Use the buffer inlet syringe to manually move the worms into the channels, and the outlet syringe to reverse the worm movement as necessary.
Loading the animals requires some coordination. Use gentle pushes on the syringe plungers to align along with the channel and a somewhat stronger push to drive the worm all the way in. Then, let worms adjust to their new environment for two to three hours before beginning an experiment.
The worms are confined to their channels, but they are not immobilized. Most worms will stay in their channels for the duration of the experiment, but some may escape or get entangled in the microfissures. Here, bright field images of a single, representative worm over the course of ten hours in the device can be observed.
Using transgenic worms that express GFP under the immune response gene one promoter, upon P.Aeruginosa, PA14 infection, GFP expression is observed in the midbody and tail of the worm over the first five hours of exposure to the pathogen, increasing to a robust, full-body expression over the next five hours. Plotting the total fluorescence of each worm as a function of time post-infection illustrates the dynamics of immune response gene one induction as well as the worm to worm variability in the timing and extent of induction. Time lapse imaging of the eggs laid by the worms in each channel allows plotting of the cumulative number of eggs laid by each worm as a function of time post-infection and captures the animal to animal variability as well as the pathogen-induced decline in egg-laying as the infection progresses.
Once mastered, the microfluidic device can be prepared and loaded in one hour. Once the device is loaded and the worms are acclimated, other reagents such as specific drugs or chemical cues of interest can be delivered to the worms to answer additional questions about the control of nematode neuronal and genetic systems. After its development, we and our collaborators used this technique to dissect nematode feeding behaviors as well as worm genetic responses to heat-shock infection and starvation in a variety of environments.
After watching this video, you should have a good understanding of how to use a microfluidic device to perform longitudinal essays.
