Figure 6.
Fibrin deposition around TNF-α–treated endothelial cells under flow conditions. (A) Engineered microvessel setup. HUVECs were grown to confluence on engineered microfluidic substrate and pretreated with TNF-α (100 ng/mL), overnight (B-C) or for 4 hours (D-H). The cell layer was stained with CellMask Orange or Deep Red to visualize the plasma membrane (pseudocolor blue B, C; magenta D-H). Fresh plasma (B, C) was supplemented with corn trypsin inhibitor and fluorescein-labeled fibrinogen (green) and passed through the microfluidic with a sheer of 500 seconds−1 for 30 minutes. The microvessels were imaged with fluorescence microscopy. (B) Representative image of fibrin definition in TNF-α–treated microvessel perfused with plasma at 500 seconds−1. Fibrin deposition was not observed in the control channel and was prevented by lactadherin in TNF-α–treated microvessels. (C) Enlarged view of TNF-α–treated microvessel with arrows identifying fibrin deposition at cell junctions or intercellular gaps. (D) Fibrin deposition was also evaluated after perfusion of fresh whole blood, supplemented with corn trypsin inhibitor and fluorescein-fibrinogen at sheer of 100 seconds−1. Fibrin accumulated at cellular junctions (magenta pseudocolor) in a reticular pattern (magenta) as well as in strands in the direction of flow. (E) Enlarged view of boxed region in panel D with fibrin staining shown as white in the top panel, membrane staining as white in the bottom panel, and merged image in the middle. Arrows highlight junctional deposition of fibrin coincident with intact junctions (cyan) or gaps containing junctions with filament-/filopodia-like structures (yellow). (F) Representative field of view under conditions as in panel D, at higher magnification. (G) Enlarged view of boxed region in F with fibrin shown in white (left) membrane in white (right) and merged (middle). Arrows (G-H) highlight junctional deposition of fibrin coincident with intact junctions (cyan) or gaps containing junctions with filament-/filopodia-like structures (yellow). Asterisks indicate occasional fibrin depositions initiated at discrete locations on the cell body. (H) Further enlargement of the boxed region in panel G.

Fibrin deposition around TNF-α–treated endothelial cells under flow conditions. (A) Engineered microvessel setup. HUVECs were grown to confluence on engineered microfluidic substrate and pretreated with TNF-α (100 ng/mL), overnight (B-C) or for 4 hours (D-H). The cell layer was stained with CellMask Orange or Deep Red to visualize the plasma membrane (pseudocolor blue B, C; magenta D-H). Fresh plasma (B, C) was supplemented with corn trypsin inhibitor and fluorescein-labeled fibrinogen (green) and passed through the microfluidic with a sheer of 500 seconds−1 for 30 minutes. The microvessels were imaged with fluorescence microscopy. (B) Representative image of fibrin definition in TNF-α–treated microvessel perfused with plasma at 500 seconds−1. Fibrin deposition was not observed in the control channel and was prevented by lactadherin in TNF-α–treated microvessels. (C) Enlarged view of TNF-α–treated microvessel with arrows identifying fibrin deposition at cell junctions or intercellular gaps. (D) Fibrin deposition was also evaluated after perfusion of fresh whole blood, supplemented with corn trypsin inhibitor and fluorescein-fibrinogen at sheer of 100 seconds−1. Fibrin accumulated at cellular junctions (magenta pseudocolor) in a reticular pattern (magenta) as well as in strands in the direction of flow. (E) Enlarged view of boxed region in panel D with fibrin staining shown as white in the top panel, membrane staining as white in the bottom panel, and merged image in the middle. Arrows highlight junctional deposition of fibrin coincident with intact junctions (cyan) or gaps containing junctions with filament-/filopodia-like structures (yellow). (F) Representative field of view under conditions as in panel D, at higher magnification. (G) Enlarged view of boxed region in F with fibrin shown in white (left) membrane in white (right) and merged (middle). Arrows (G-H) highlight junctional deposition of fibrin coincident with intact junctions (cyan) or gaps containing junctions with filament-/filopodia-like structures (yellow). Asterisks indicate occasional fibrin depositions initiated at discrete locations on the cell body. (H) Further enlargement of the boxed region in panel G.

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