The CApillary FEeder (CAFE) Assay: A Method to Track Food Consumption and Preference in Drosophila
筆記録
– To prepare for the Capillary Feeder, or CAFE assay, insert graded or marked glass capillaries filled with colored, liquid food, bottom end first, through the pipette tips on top of a preassembled experimental vial. Ensure the capillaries are securely placed at the same height and are not で contact with the apparatus.
Mark the starting level of the meniscus で each capillary. Then house the vials で a temperature-, humidity-, and light-controlled environment for the duration of the experiment while replacing capillaries as needed. During the CAFE assay, mobile flies are unrestrained and have free access に the bottom open end of the micropipette. Flies feed voluntarily as they extend their proboscis and intake the liquid, reducing the medium inside the capillary.
To track food consumption and preference for a set time period, record the descent of the meniscus from the starting mark に the level at the end of that period from qualifying capillaries. In the example protocol, we will see the CAFE assay with the assembly modified に achieve reduced evaporation from the liquid-filled capillaries.
– 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 に the capillary. Repeat this step for all food solutions tested.
Next, place the capillary ends inside all of the vials at the same level, 3 to 4 centimeters from the lid. Leave some distance between the filter paper に prevent the capillary from leaking. Use a marker pen に label the upper end of the colored liquid. Label them individually using a stripe code に ensure the different capillaries can be identified.
Place prepared CAFE assays into a plastic box with a gridded inlay. Use a sponge bung に apply 100 microliters of fresh water every 24 hours に 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 に the CAFE assays で the plastic box.
Cover the plastic box に create a humidity-controlled environment during the experiment. Transfer the box に a secure position で 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 に calculate the consumption per fly for the following period.
At the end of the assay and before replacing the capillary, keep the CAFE assay で 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 で 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 measurement.
