Our protocol is designed to perform a fine assay for the fruit fly, which is a powerful model organism that enables to design for all the genes, receptors, neurons and a neuro-psych heads that are involved in test sensation and food-consumption. Like humans fly like food that tastes:sweet, slightly-salty, slightly-sour or reject food that tastes bitter, too salty or too sour. The power of this technique comes from the speed and convenience.
You can test many different flies in a short time PRH without a complex setup. Our method is an excellent, excellent assay for understanding whether flies like a, just like a particular food, as well as how certain gene products influence taste preference. Establishing whether animals accept or reject certain foods can be challenging.
Therefore it's important to establish a standard protocol for testing animal taste preference. To prepare starvation vials for the assay, loosely compact a piece of tissue at the bottom of each empty plastic fly vial per fly gene type to be tested. Such that the paper fills this space without forming a dense mass, at about three milliliters of pure water to each vial to completely saturate the tissues without standing water.
Taking care that there are no large droplets of excess water on the walls of the vials. Alternatively five milliliters of 1%Agros without sucrose can be substituted for the soaked paper, 24 hours before the experiment carbon dioxide anesthetized two to four day old flies and sort the flies into groups of approximately 70. Then add each group of flies to a starvation vile and label each vial with the genotype and time of starvation initiation.
Before performing an experiment, prepare a range of dilutions for each dye using the same food with each different dye color. Combined 0.5 grams of Agros with 50 milliliters of pure water in a microwave safe vessel and heat the Agro solution until it has dissolved stirring as necessary. Dissolve each food component, including sucrose and any experimental compounds in water at a 100 fold or higher concentration of the final tested concentration.
And mix the Agro dye and desired experimental compound in conical, polypropylene centrifuge tubes. After mixing place the tubes in the 60 degrees Celsius water bath until distribution and add one milliliter of one color of the experimental food and dye solutions to one side of the first assay dish. Allow the Agros to cool until firm before adding one milliliter of the control food solution, labeled with the second dye color to the other side of the dish.
Then repeat the assay dish preparation, with the opposite control and experimental dye solution pair. Use the results to identify two dye concentrations that yield a preference index of zero. When no experimental compound is added.
To perform a two-way feeding assay. First temporarily paralyzed one experimental fly vile for three to five minutes on ice until no obvious motor activities such as flying and climbing are observed. Once the flies have been immobilized, gently invert the vial and tap to transfer the flies into the assay chamber then quickly placed the lid onto the chamber.
When all of the groups have been transferred move all of the assay chambers to a dark and closed space for 90 minutes. At the end of the assay invert the chambers at a minus 20 degrees Celsius freezer, for about an hour. Before placing each chamber under a stereo microscope to examine the abdominal color of the flies in each experimental group.
Count a fly, if the abdomen is more than 50%colored indicating a robust feeding, discard any flies in which the abdomen contains only a tiny food spot, which is indicative of poor eating. When all of the flies have been counted use the equation to quantify the food preference index for each group. In this assay, a 35 millimeter dish was divided into two week we'll feed in compartments containing Agro's food, coupled with red or blue dye.
To exclude dye bias, the blue and red dye concentrations were carefully refined to yield an approximate zero preference when only these two dyes were added, 90 minutes after wet starved adult fly loading bly abdominal colors appeared as blue or red. If the animal predominantly consumed blue or red food, respectively, if a fly consumed both blue and red, the abdomen appeared purple. Only flies that ingested a considerable amount of food were scored, while flies with an insufficient food intake were not counted.
Comparing this Petri dish based assay to the multi-well plate based assay revealed that the two feeding methods gave essentially the same assaying feeding results in response to sweet, bitter, and salty food in wild type flies. Notably, however it is much faster to prepare and distribute food in a Petri dish than in a 60 well multi-well plate. Be sure to identify what dye concentration to use especially after making a new diet batch.
This concentration can be different between fly lines. Our pair today is based two-choice assay is very powerful too. For you asking how the flies, sense both internal-external frame damaged to real state appropriate feeding and behavior.
