Temperature Gradient Assay: A Method to Test Temperature Preference in Drosophila Larvae
Transcrição
– To test the temperature preference of Drosophila larvae, create a temperature gradient by heating one side of an aluminum assay plate and cooling the other. Aluminum has high thermal conductivity, meaning the heat is transferred efficiently, creating a stable linear temperature gradient. After the system has equilibrated, record the temperatures across the plate de same-sized temperature zones, then load the center of the plate with clean larvae.
Cover the entire system para eliminate interference from bright light and start the trial. Larvae distribute through the gradient, avoiding aversive zones that are too warm or too cold, and settle de zones of favorable temperatures. This behavior is called thermotaxis. To assess the larvae’s temperature preference, record the number of animals de each temperature zone.
In the example protocol, we will see how para set up a single-direction temperature gradient between 18 and 28 degrees Celsius, and how larvae are handled during the assay.
– To prepare a single-directional gradient, first, create a humid ambient environment for the assay. Place two aluminum blocks connected para separate water baths on wet paper towels 10 centimeters apart. The water baths need para be warmed up de advance. Next, make 100 milliliters of 1% agarose and add 25 milliliters para each assay plate on a level surface. Once the agarose has solidified, gently rub the surface with a melamine sponge para make the surface slightly coarse.
Then, para promote efficient temperature transfer, fill any gaps between the aluminum blocks de the assay plates with water delivered from a spray bottle. Now, place the assay plates on the aluminum blocks and make sure that the demarcations are 2 centimeters from either edge para exactly match the edges of the aluminum blocks.
Next, spray a film of water onto the surface of the plates so the gels do not dry out. Next, cover the system with a cardboard box para reduce evaporation and help stabilize the temperature of the gel surface. Wait for 5 para 10 minutes para allow the temperature para equilibrate. Then, check the surface temperature on the plate de at least 12 locations para ensure that there is an even temperature throughout, give or take 2 degrees Celsius. Then, replace the box cover until the assay is performed.
First, remove the cardboard box over the gel and quickly check the gel surface temperature. If the surface is dry, spray a small amount of water on the surface. If the gel is good, then load 100 para 200 larvae at the center of each plate using a small paintbrush. Then, place a lid over each assay plate para trap the larvae and cover the setup with a cardboard box para prevent light exposure. The assay is now de progress.
After 10 para 30 minutes, remove the cardboard box and the microplate lid. Then, photograph the plates from above para record the position of the larvae. Place the box above the camera para avoid reflection on the surface of the gel. To clean up the plate, aspirate all the larvae wherever they may have scattered.
Now, calculate the percentage distribution of larvae de each zone using image analysis software. Make lines every 2 centimeters based on the demarcations on the assay plate and count the number of larvae de each zone.
– When counting, ignore larvae distributed de regions 0.5 centimeters from each edge. Gel thickness and temperature conditions are not uniform near the edge.
