A medição da taxa metabólica em<em> Drosophila</em> Usando respirometria

The goal of this procedure is to determine the relative metabolic rates of flies by measuring their carbon dioxide production. This is accomplished by first placing the flies into specially prepared respir barometers. Next, the Respir barometers containing flies are inserted into a chromatography chamber making contact with a colored aqueous solution.

Over time, the liquid migrates into the respirometer due to decreasing pressure caused by the removal of expelled carbon dioxide from the gas environment before and after. Images of the experiment are used to calculate the amount of carbon dioxide produced based on the dimension of the respirometer capillary. Ultimately, the data obtained in this assay will allow the comparison of the relative metabolic rates between experimental and control flies.

To begin, cut a 1000 microliter pipette tip with a razor blade as straight as possible, and insert the micro pipette into the 1000 microliter pipette tip. Next, place a piece of foam into the pipette and push it down. Then add a small amount of soda lime and secure it with a second piece of foam.

Apply glue where the micro pipette is inserted into the pipette tip and allow the glue to dry overnight. Prepare the chamber solution by mixing water with eosin in a one to five ratio, which will result in visible colorization. Next, pour the ees and water solution into the chamber.

Then label the vertical side of the chamber with the centimeter scale and the horizontal side with the respirometer labels. Prepare the respir barometers by labeling them individually with a marker. If you are analyzing several genotypes, do not forget to make a note of the respirometer number and corresponding genotype.

Next immobilize flies using a pre-cool metal plate and place three to five flies of the desired genotype. Inside each respirometer seal the respir barometers tightly at the top using plasticine putty and allow the flies to recover from an anesthetization for 15 minutes. Prepare one respirometer without flies to be used as the atmosphere control.

Next, attach a 1.5 milliliter einor tube holder. Open on both sides at the top of the chamber and hang the respir barometers in the chamber with the tips submerged in the colored solution. Add Vaseline between the lid cover and the chamber to provide stronger isolation from temperature and pressure fluctuations.

Next, close the lid and allow the system to equilibrate for 15 minutes. Then take a photograph of the chamber, making sure that the level of liquid within each micro pipette and the scale are in frame. After one to two hours, take another picture To begin analysis.

Open acquired images using Image J software. Next, set the pixel scaling in the software. Using each picture as a reference, then measure the distance or delta D, that the liquid traveled from a determined reference spot in images taken at the beginning and end of the experiment.

Finally calculate the amount of carbon dioxide produced. Using this method, it was observed that the flies carbon dioxide production positively correlates with housing temperature. These results contrast with District Li Ccan or DG mutants whose carbon dioxide production significantly increased compared to wild type flies.

The described above method allows primary identification of genetic mutations in flies that can lead to changes in metabolism.