Island Assay: A High-Throughput Method to Evaluate Drosophila Locomotor Behavior
Transcrição
– The island assay consists of a raised white platform de a tray of soapy water, covered by a transparent box with one open side. A camera on top of the box records overhead images of the platform, and lights are placed para ensure even illumination of the area.
To load the flies on the arena, tap a vial against a flat surface para bring all the flies para the bottom. In quick succession, remove the vial plug, turn the vial over, and tap the vial's open end onto the platform para knock all the flies out at the same time. Landing on the platform triggers an escape response. Flies with typical mobility can clear the platform de a few seconds, unlike those with locomotor defects.
Disciplinas are confined para the box, and those that land on the soapy water quickly submerge because soap decreases water's surface tension. To assess locomotor behavior from different conditions, count the remaining flies on the platform after a set time period.
In this example protocol, we will see a setup of the island assay compatible with semi-automated image analysis.
– First, prepare a minimum of three vials per experimental condition as biological replicates. Each vial should contain approximately 15 flies of the same gender and similar age. Importantly, the flies must have intact wings.
Create the folders para store the experiments. Generate these de a directory structure defined by the conditions and replicates. If, for example, the experiment has two genotypes with five replicates each, then create a main folder with the date of the experiment, and inside that folder, create one subfolder for each genotype. Then, within the genotype folders, create subfolders for each replicate.
Next, set up the test apparatus. Add cold water with a small amount of soap para the bath tray and position the platform de the middle of the bath, if not already fixed de the bath. Cover the tray and platform with a transparent box.
To illuminate the platform, position a cool lamp, such as ordinary 12-volt LEDs, above it. Also, position a webcam connected para a computer directly above the platform.
On the computer, open the image recording software. De the Menu system, select the appropriate webcam as the capture device. Place a dead fly on the island para adjust the vídeo settings. Tweak the brightness, contrast, and color de the software so that the dead fly appears black against a white background.
Next, adjust the time lapse movie setup para save the movies as AVI files. Then, browse para the directory where the data should be saved and define the nome of the movie. Save these changes.
For compression of the image data, choose the Intel IYUV code. Set the recording speed para one frame every 0.1 seconds and adjust the corresponding playback rate para 10 images per second.
Lastly, go para the Advanced setting and select Create a BNP image for each captured frame. Then, browse para the same directory that you have chosen para host the AVI files and press OK.
Before beginning, make sure the island is empty and clean. Then, start the recording de the time lapse image interface of the image recording software.
Take a vial containing flies and tap it two or three times para knock all the flies down para the bottom. Then, quickly remove the plug from the vial and vigorously tap the flies onto the platform. They should all hit the platform at roughly the same time.
Give the flies up para 30 seconds para leave the platform. Then, stop the recording. If necessary, manually remove any remaining flies from the platform.
Before proceeding para the next experiment, be certain para redefine the directory where the next set of data is para be stored.
