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Blood, Vol. 103, Issue 9, 3388-3395, May 1, 2004
Granulocyte transmigration through the endothelium is regulated by the oxidase activity of vascular adhesion protein-1 (VAP-1)
Blood Koskinen et al.
103: 3388
Supplemental materials for: Koskinen et al, Vol 103, Issue 9, 3388-3395
Files in this Data Supplement:
- Video 1. Transmigration of polymorphonuclear leukocytes (PMNs) through a monolayer of human umbilical vein endothelial cells (HUVECs) (Video, 4717 KB)
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The video shows transmigration of PMNs through a monolayer of VAP-1 transfected HUVECs under a constant shear stress of 1.0 dyn/cm2. Surface-adherent PMNs are phase-bright, whereas the transmigrated cells are phase-dark (all noninteracting leukocytes have already been flushed away at this point; see “Patients, materials, and methods” for details). HUVECs are the dark large cells that form a confluent monolayer at the bottom of the capillary. A sequence of events during a period of 4 minutes and 40 seconds is shown at 20 times the original speed. In the original video, 25 frames were collected per second; in this output, the frames/second display rate is 15. The 3 white arrows that appear in the video point to representative transmigrating PMNs. At the end of the video, each of the 7 transmigrated PMNs is indicated by a white arrow. The rest of the leukocytes are phase-bright surface adherent PMNs, which can also actively migrate on the top of the HUVEC monolayer. Thus, the transmigration percent in this particular field is 13.5% (7 transmigrated cells/52 interacting cells [7 transmigrated cells + 45 surface-adherent cells]). Bar, 20 µm.
Methods
HUVECs transfected with vascular adhesion protein-1 (VAP-1) were grown to confluency at the bottom of a perpendicular class capillary, and freshly isolated PMNs were perfused over the monolayer of these endothelial cells using laminar shear stress 1.0 dyn/cm2 as in a typical transmigration assay. The leukocytes were perfused for 5 minutes, and thereafter only the binding buffer (without the cells) was perfused with the same constant shear rate, using a computer-controlled syringe pump, for the rest of the experiment. The whole experiment was recorded in real time using phase-contrast optics (LCPlanF1 20x, NA 0.40), black-and-white video camera, and digital videos as detailed in the paragraph above. Starting from t = 5 minutes 26 seconds (t = 0 being the time when the perfused leukocytes appear in the microscopic field), a 4-minute-and-40-second segment of the video is shown at 20 times the original speed. The selected video segment was captured and edited (the speed was adjusted and the arrows were added) and compressed (using Cinepak Codec by Radius; Oakland, CA) using Adobe Premiere 5.1 (San Jose, CA).
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