This study investigates the dynamics of immune cell migration during epithelial wound healing in transgenic Drosophila. Using confocal microscopy, the behavior of hemocytes in response to chemoattractants released from wounded cells is analyzed.
Begin with a glass-bottomed dish containing transgenic Drosophila pupae carrying labeled wounded epithelium with their cell surface proteins exhibiting green fluorescence.
The pupae's immune cells — or hemocytes express photoconvertible green fluorophores in the cytoplasm and red fluorescent proteins in the nucleus, facilitating cell tracking.
The wounded cells release chemoattractants — or inflammatory molecules, attracting the hemocytes to the wounded site.
Confocal microscopy reveals the wounded area encircled by green epithelial cells.
During healing, green hemocytes with red nuclei near the wound extend membrane protrusions — filopodia and lamellipodia and migrate toward the wounded area.
Over time, as the chemoattractant diffuses, distant hemocytes migrate toward the wound, creating an immune cell wave.
Illuminate the subpopulation of migratory hemocytes with a 405-nanometer wavelength. This irreversibly converts the hemocytes' photoconvertible fluorophore from green to red.
These photoconverted hemocytes repair the wounded epithelium and move away from the wound site, differentiating the photoconverted hemocytes from non-photoconverted hemocytes, and elucidating inflammatory cell dynamics.
After wounding the pupil wing margins, quickly transfer the glass-bottomed dish to an appropriate microscope for time-lapse imaging. Then, open the appropriate image capture software.
In the software, turn on the appropriate lasers, and adjust their power and gain offset to get enough fluorescent signal without pixel saturation. Generally, the lowest possible laser power in the range 5% to 20% works best to minimize photobleaching.
Focus on the whole pupil wing under low magnification or focus on the wound under high magnification to investigate wound repair. To capture both the repairing epithelium and inflammatory cell recruitment, first, set the microscope to record a z-stack using the fine focus adjustment on the control panel. Scan from the wounded epithelium through to the extracellular space beneath, containing migrating hemocytes.
Next, set the software to record z-slices through the pupil wing every 3 microns or at even tighter intervals. For time-lapse imaging, record z-stacks at least every 30 seconds for at least one hour.
When using the photo-convertible probes to selectively photo-convert and label a subset of cells during imaging, open the appropriate modules within the imaging software to perform the photo-conversion and activate the 405-nanometer laser. Then, select the cells to be photo-converted within the FRAP software using a selection tool.
Next, set the time course for photo-conversion to a single iteration frame, and set the 405-nanometer laser to 20% laser power, then click Start The Experiment to perform photo conversion. Once completed, exit the FRAP module and return to the original imaging screen. There, tune the lasers to the fluorophores in use and image the photo-converted and non-photo-converted cells using time-lapse recordings.
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