This method is used to evaluate the crop motility through counting crop contraction and detecting the food distribution in just the digestive. The advantage of this technique is that it's continually evaluates crop motility. It's low cost and easy to perform.
This method makes it possible to use just filler as a model to study digestive function on the food passages in the gastrointestinal tract. Helping to demonstrate the process will be Junmeng Xi, a graduate student from my laboratory. Maintain flies in vials containing 10 milliliters of freshly made food in a 25 degree celsius incubator with 60%humidity and a 12 hour light, 12 hour dark cycle.
Ensure that a large number of the desire phenotype flies eclose simultaneously by culturing young flies with standard food with dry yeast then transfer the adult flies to a vial with wet yeast and allow two days for egg laying. Leave the eggs in the incubator to develop and transfer adult flies to a new vial to collect more eggs. Collect the eclosed male or female flies each day and culture them in new vials with standard food to the desired age.
Anesthetize the flies with carbon dioxide and transfer one fly into a dissecting plate well with 200 microliters of PBS. Grasp the fly at its thorax with a pair of tweezers and open the thorax with another pair of tweezers then pull the ends in opposite directions to open the abdomen. Carefully take the crop and gut out of the body and wait for the fly to wake up.
When the fly is awake, count the number of times the crop contracts in one minute. Repeat the contraction counting five times, waiting 30 seconds between each count. Weigh the blue dye and dissolve it in PBS at a 20%concentration.
Then stir it into the boiled liquid maintenance food to a final concentration of 5%during the food cooling process. After keeping flies in vials with starvation food for four hours, transfer them into new vials with the dyed food and culture them for the desired time, keeping in mind that at maintenance conditions the food passes through the fly in about two hours. Transfer an anesthetized fly into a dissecting plate well containing 200 microliters of PBS.
Use tweezers to grasp the fly at the thorax and remove the head with another pair of tweezers, then transfer the body to a new well with PBS and wash it with gentle shaking to remove the dye attached to the body. Open the abdomen with two pair of tweezers and carefully separate the whole gut from the body. Take the crop off of the whole gut and put it in a tube with 100 microliters of PBS.
Put the rest of the gut in another tube with 100 microliters of PBS then use pipette tips to grind the crop and gut and dissolve the dye in PBS. Repeat the dissection until enough crops and guts are collected. After collecting the samples, centrifuge the tubes at the highest speed for one minute and transfer 90 microliters of supernatant to well of a 96 well plate.
Make a series of standard blue dye dilutions at concentrations from one times 10 to the 7 to 10 to the 4th grams per milliliter and add them to the wells. Measure the absorbance at 630 nanometers of the wells and use the standard measurements to plot a line graph of absorbance vs dye concentration. Then use the curve to calculate the dye concentration of the samples.
The crop motility assay was used to count crop contractions on three day old NPRL mutant flies and genetic background controls. The mutation was found to significantly decrease the rate of crop contraction. To further confirm the function of NPRL2 on the crop, food movement was detected by feeding dyed food to the flies.
All flies had similar amounts of dye in the crop but the NPRL2 flies had less dye in the gut which may be associated with the decreased crop contraction rate. When typing this protocol, make sure that the crop remains contact to the body and the fly is alive and awake. Otherwise, the crop will lose the ability to contract.
During the dissection process, the crop and the gut should be in contact. If dye leaks into the dissection medium, the sample cannot be used anymore. Let's just fill our appetite and the food ejection within a short time can also be evaluated by detecting the dye amount in the whole gut.
