PMNs are activated following a laser-induced injury on the cremaster. (A) PMNs isolated from WT mice were loaded with Fluo-4-AM. Exogenous PMNs (3 × 106 cells) were infused into the circulation of a recipient WT mouse. Following a laser-induced vessel wall injury, thrombus formation was observed and images recorded over time. Representative composite images of fluorescence and brightfield data depicting thrombus formation showing calcium mobilization in Fluo-4-AM–loaded PMNs (green) in a WT mouse (representative of 30 thrombi performed in 3 mice). (B) Confocal imaging of calcium mobilization in Fluo-4-AM–loaded PMNs (green). The solid line indicates the boundary of the vessel and the dotted line indicates the boundary of the thrombus. (C) Representative pictures showing the visualization of neutrophil elastase activity in the bloodstream of a WT mouse at the site of a laser-induced injury before (upper panel) or after (lower panel) depletion of PMNs. Neutrophil elastase activity is depicted in yellow. Graphs represent the medians of neutrophil elastase-integrated fluorescence intensity (left) and the maximal-integrated fluorescence intensity of neutrophil elastase (right) in WT mice in presence (32 thrombi in 3 mice) or absence (32 thrombi in 3 mice) of PMNs. *P < .05.
