Real-time adhesive capture of a circulating blood cell cluster at a Drosophila third-instar larval wound site. Real-time movie of blood cells visualized from dorsal side of a Nrg-GFP;PxnYFP larva. A circulating blood-cell cluster flows from the larval anterior to the wound site (area outlined in white in first and last frames), where it is captured from circulation. Note that after docking at the wound site, the cluster pivots around a single tether point for several seconds before attaching more securely to the wound. This cell cluster remained firmly attached to the wound site for another 30 s of filming (not shown). Red circle in first frame indicates where the cluster first comes into view and red circle in final frame indicates docking site in the wound. Time between frames, 0.325 s. Total duration, 26 s. Fig. 3A was generated from individual frames of this movie. Movie is Movie S10 of Proc Natl Acad Sci (2008). 105: 10017-10022. Movies from this publication include CIL #s 25607, 25608, 25609, 25610, 25611, 25612, 25613, 25614, 25615, 25616, 25617.
Third-instar [w, Nrg-GFP;Pxn-Gal4, UAS-2xeYFP] larvae were wounded and mounted dorsal side up on a glass slide with the anterior and posterior ends of the larvae taped down to varying degrees to prevent locomotion and constrain body peristalsis. Larvae were imaged on a Leica MZ16FA fluorescent stereomicroscope using a Planapo 1.0× objective, and images were captured on a monochrome Leica DFC350FX digital camera. For real-time recording, frames were captured every ≈400 ms over a 4- to 40-s period. Images were obtained by using the Scope-Pro Advanced Acquisition plug-in, and image analysis was carried out with Image-Pro AMS ver. 5.1 Software (Media Cybernetics). The object-tracking tool was used to manually track specific blood cells in a time series. Velocities of tracked cells were calculated by using coordinates provided by the software.
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