毛细管馈线分析测量的摄食

The overall goal of the CApillary FEeder assay is to observe and measure liquid food intake of adult flies. This method can help to answer key questions in metabolic research such as how actual feeding behavior is initiated and how food consumption is terminated. The main advantages of this technique are that it's easy to use and highly reliable.

Visual demonstration of the method is critical as several preparatory steps need to be carefully executed for consistent results. To begin the experiment, obtain a drosophila melanogaster culture plastic vial and seal it with a manufactured Plexiglass lid containing an O-ring. Use five microliter microcapillaries with one microliter marks to present the liquid food.

Cut off the top of a two to 20 microliter pipette tip and insert the tip into the whole of the lid to position the capillaries in the conical openings. Insert an uncut two to 20 microliter pipette tip into the same opening to prevent flies from escaping. Load eight or 20 flies by trapping or with a blow pipe through the lid's central opening and close the hole with a sponge bung.

Next, place the multiple prepared vials into a plastic box with a gridded inlay to handle them safely. To control breeding conditions, introduce 35 virgin females and 15 males for each experimental group into a plastic culture vial containing 50 milliliter fly food. Allow the flies to lay eggs for the first three days, then transfer adult flies to fresh food vials and let them lay eggs for two more days.

After two days, repeat the transfer. On day seven, discard the adult flies. Next, anesthetize two to three day old adult flies using a CO2 pad and collect 100 flies sorted by sex in a 1.5 milliliter plastic tube.

Measure their weight with a standard laboratory scale. Determine the wet weight of at least four independent fly groups sorted by sex and use the weight to calculate the microliter food consumption per milligram fly. For a three hour assay, use 20 flies in two filled capillaries.

For a long term experiment, use a group of eight flies with a supply of four filled capillaries. Fill up to 10 capillaries simultaneously with previously prepared colored food solution. Insert the capillaries into the food solution and then tilt them to a 45 degree angle to accelerate filling.

Stop when the liquid reaches the top five microliter mark of the capillary. Remove excess solution on the outside and inside of the capillary with tissue paper. Carefully remove the pipette tip that is closing one of the outer openings and insert a filled glass capillary, bottom end first.

Secure the capillary by placing the pipette tip back next to the capillary. Repeat this step for all food solutions tested. Next, place the capillary ends inside all of the vials at the same level three to four centimeters from the lid.

Leave some distance between the filter paper to prevent the capillary from leaking. Use a marker pen to label the upper end of the colored liquid. Label them individually using a stripe code to ensure the different capillaries can be identified.

Place prepared CAFE assays into a plastic box with a gridded inlay. Use a sponge bung to apply 100 microliters of fresh water every 24 hours to keep the humidity constant inside the assay. Use four separate vials filled with 30 milliliters of double distilled H2O as humidity devices and place them next to the CAFE assays in the plastic box.

Cover the plastic box to create a humidity controlled environment during the experiment. Transfer the box to a secure position in a temperature, light, and humidity controlled climate chamber for the experimental period. Before each 24 hour interval, make note of the dead flies and use the number of live flies to calculate the consumption per fly for the following period.

At the end of the assay and before replacing the capillary, keep the CAFE assay in the upright position. Do not remove the lid and mark the lower meniscus of the capillary. Discard the data if the mark end is not below the initial mark.

Replace the used capillaries with freshly filled capillaries for long term experiments every 24 hours. Measure the decline of the meniscus, then discard the old capillaries. Then carefully remove the capillaries from the assay and store them for data collection.

Check if the liquid inside the capillary reached the lower end. If the liquid inside the capillary did not reach the lower end, discard the data. After the experiment, dismantle the setup and wash the vials, lids, and sponge bungs in a soap bath.

Then dry them overnight at room temperature for further use. Repeat experiments with the same genotypes on at least three different days. Using a caliper, measure the distance between mark beginning and mark end on the capillary.

Finally, discard the capillaries after the measurements. Two food choices were offered, a 1 molar sucrose solution and a 1 molar sucrose solution with 15%ethanol. Visual examination of the abdomen indicates the flies feed on both solutions.

However food consumption per fly is nearly two folds greater for the solution containing ethanol. The ethanol preference was also observed in long term experiments where a group of eight flies had access to similar food sources for four days. The CApillary FEeder assay also reveals that male and female flies prefer higher sucrose concentrations over lower sucrose concentrations.

And male flies consume significantly more of the high sucrose concentrations than their female counterparts. While attempting this procedure, it's important to remember to perform the assay in a controlled fashion and to repeat the experiment with the same genotypes on at least three different days. In addition, when testing mutant flies, it's important to consider whether the flies can actually perform the task required to obtain the food, such as sending the food source or climbing up and down the capillary.