The overall goal of this assay is to determine any changes in fat storage levels that may be caused by a change in genetics, diet, or treatment. These methods can help answer key questions in the field of metabolism such as genetic or neuronal contributions to obesity predisposition. The main advantage of this technique is that it's fast, economical, and very reproducible when compared to other methods currently used.
Individuals new to this method or new to working with wandering stage larva may struggle with this protocol because the developmental timeline of different genotypes can be highly variable. To begin this procedure, add a small dollop of yeast paste to the middle of a grate plate. Smooth down any rough edges with a spatula.
Next, transfer the flies of interest into an egg collection chamber, and place the grate plate with yeast paste on top facing inwards. Then tape the grate plate to the egg collection chamber to keep it from falling out. Make sure to write important genotype and date information on the bottom of the grate plate.
Subsequently, upend the egg collection chamber to allow the flies to lay eggs on the grate plate. After that, cover the egg collection chamber with an open box and place the covered egg collection chamber in an incubator at 25 degrees celsius for four to six hours to allow the flies to lay eggs. When it is done, take the grate plate out of the egg collection chamber and transfer the flies back into their original bottle of food.
Use a spatula to wipe off any excess yeast paste from the grate plate. Subsequently, store the grate plate in a larger Petri dish in a humidified incubator at 25 degrees celsius for 24 hours. In this procedure, score the top layer of the food by plunging it with a spatula several times to allow the larvae to burrow and feed.
22 to 24 hours after egg collection use a small paintbrush to collect 50 first instar larvae of the same approximate size and place the larvae in the experimental food vial. It's very important that larvae of the same approximate size are selected and transferred to the experimental food. This will ensure tight coordination of larval development.
Next, clean the paintbrush with a laboratory wipe and ensure that there are no remaining larvae on the paintbrush before continuing to another genotype. Afterward, place the vial of larvae in an incubator at 25 degrees celsius and allow them to develop. Remove the vial of larvae from the incubator once there are 20 to 40 larvae wandering up the sides of the vial.
Record the date and time of the assay as well as the number of wandering larvae. Then prepare the experimental solution by adding 11.5 milliliters of PBS and nine milliliters of 20%sucrose to a 50 milliliter conical tube. Add 20%sucrose to the vial until it reaches one inch from the top of the vial.
Use a spatula to gently stir the food top layer to free the larvae that are still burrowing in it. All the larvae will rise to the surface of the sucrose solution. Afterward, gently transfer the larvae into the initial sucrose solution and gently stir them.
While transferring the larvae to the experimental solution, be careful not to squeeze or puncture the larvae. This may alter the results of the assay. Cap the conical tube and upend it several times to thoroughly mix the solution.
Then uncap the conical tube and swirl it to create a gentle vortex. Allow two to five minutes for the larvae to settle, either floating to the top of the solution or sinking to the bottom. Record the number of floating larvae, and the specific sucrose concentration tested.
Then add one milliliter of 20%sucrose to the experimental solution to increase its density. Subsequently, use a spatula to stir the larvae gently. Following this, cap the conical tube and upend it several times to thoroughly mix the solution.
Subsequently, uncap the conical tube and swirl to create a gentle vortex. Record the number of floating larvae and the new concentration. Continue this process until at least 95%of the larvae are floating.
Add 20%sucrose to the conical vial until it is one inch from the top. Next collect all the larvae in a dissection dish filled with PBS. Examine the larvae under a microscope for any small larvae, prepupae, pupae, or any other differences between genotypes to ensure that the procedures were done appropriately.
Then record the total number of larvae. Afterward, transfer 10 larvae on a laboratory wipe for drying. Label a microcentrifuge tube and place the larvae in the tube.
Subsequently, flash freeze the larvae in liquid nitrogen and store the tube at minus 20 degrees celsius. Shown here is an example of how the buoyancy-based assay can detect both higher and lower fat stored in the genetically manipulated larvae. This figure shows that excitation or silencing of a certain subset of neurons in the larval brain induces lower and higher stored fat respectively.
The same genetic background control was used in both cases. Here is an example of how this phenotype obtained by the density assay is corroborated by neutral lipid extraction and quantification by GCMS. Once mastered, this technique can be done within 30 minutes if properly performed, and may be performed in tandem with other genotypes.
While attempting this procedure, it's important to remember to synchronize the development of the experimental larvae as much as possible. Following this procedure, larvae can be snap frozen in liquid nitrogen to be used in other methods like gas chromatography mass spectrometry in order to obtain additional information like the specific classes of lipid types in the larvae. After its development, this technique paved the way for researchers in metabolism to explore the genetic and or neuronal contributions to obesity in Drosophila.
After watching this video, you should have a good understanding of how to quickly screen genotypes for changes in levels of stored fats.
