Rolling neutrophils form ENDS. (A) Consecutive frames of rolling neutrophils in cremaster venules of mice. Before intravital microscopy (IVM), the mice were intrascrotally injected with TNF-α to stimulate neutrophil interaction with the vasculature. Neutrophils were labeled via i.v. injection of Ly6G-AF647 antibody. Blood flow is from left to right. Asterisks indicate ENDS-forming neutrophils, and arrows point to ENDS. Dotted lines indicate the venule wall. Scale bar, 8 µm. (B) Analysis of neutrophil rolling speed and WSS in the venules shown in A. ENDS-forming neutrophils (indicated with full symbols) had the highest rolling velocity in both examples. Red lines indicate median values. (C) ENDS (indicated by arrows) of various lengths observed in cremaster venules. Mice were pretreated as described above. Scale bars, 6 µm. (D) Human neutrophil labeled with CD16-AF647 forms ENDS while rolling on E-selectin substrate at 40 dyn/cm² WSS. Arrows indicate ENDS. Scale bar, 10 µm. (E) Image segmentation was developed to quantify ENDS formation in records of neutrophils rolling on E-selectin substrate. Cell body is color coded with cyan, tethers that are still attached to the cell are magenta, and ENDS that are not attached to the cell are yellow. Scale bar, 20 µm. (F) Human or mouse neutrophils rolled on P-selectin (blue curve) or E-selectin (red curve) substrate at increasing WSS. Surface area covered by ENDS to quantify ENDS formation. One representative example of three experiments is shown. (G) Rolling mouse neutrophils were allowed to form ENDS in a flow chamber. Only the upstream half was coated with P-selectin; the downstream half was uncoated. Neutrophils and sliding ENDS detached as they reached the noncoated half of the chamber. Upper panel shows a snapshot of rolling neutrophils and sliding ENDS. Lower panel shows kymograph analyses that were done in the positions indicated by the white lines on the upper panel. Scale bar, 50 µm. One representative example of two independent experiments is shown.