Table 1:
Assay | Application and Benefit | How to | Advantage (+) / Disadvantage (−) | Recruitment Step | Platforms or Readouts |
---|---|---|---|---|---|
In vitro flow chamber assays | - Analysis of rolling and adhesion or crawling on specific receptor ligands or endothelial cells with flow dynamics | Small rectangular glass capillaries or commercial system coated with ligands or endothelial cells | (+) detailed biomechanical analysis possible (−) isolated neutrophils |
Rolling, adhesion, spreading and migration under flow | - 20×200μm [116] or 40×400μm glass capillaries [117] - Side view flow chamber [17] - Commercial chambers with HUVEC coating [118] - Spreading under flow in commercial chambers [119] |
Autoperfused whole blood flow chamber | - Analysis of rolling and adhesion in whole blood | Canulate carotid artery of mouse and connect to coated glass capillary flow chamber | (+) whole blood including all plasmatic components (−) clotting (−) blood pressure variation |
- Rolling and adhesion | - Autoperfused glass flow chamber [44] |
Transmigration / Migration assays | - Analysis of barrier crossing with usage of defined stimuli - Pattern tracking of neutrophil chemotactic motility |
- Transwell inserts - Either only chemokine gradient or - Seeded endothelial cell layer - Possibility of combination of endothelial / epithelial cells |
(+) seeding of endothelial cells possible to mimic tissue environment (−) high variation depending on confluence of endothelial cells and chemokine gradient |
- Transmigration and chemotaxis | - Filter transmigration [120] - 3D chemotaxis [32] - Transwell with murine immortalized brain endothelial (bend5) cells [121] |
Flow cytometric assays | - Independent analysis of specific receptors - Expression vs. activation |
- Isolation of neutrophils, labeling for expression analysis or: - Activation, incubation with ligands, labeling with neutrophil markers, analysis of ligand binding |
(+) diverse applications (−) sample preparation needed and thus (−) no analysis of direct inorgan effects |
- Integrin activation and receptor expression | - Expression 121] - Activation [122] |
Neutrophil effector functions | - Assessment of neutrophil functionality | - Activation of neutrophils required - ROS: Seeding of neutrophils on coated (e.g. polyRGD) plates, stimulation, measurement of superoxide dismutase inhibitable reduction of cytochrome C by absorption on plate reader - Phagocytosis: incubation with fluorescent bacterial particles |
(+) functional relevance (+) diverse stimuli possible |
-Effector functions: Outside-in signaling (ROS), phagocytosis, NET formation | - Outside-in ROS [123] - Phagocytosis [124] |
Platelet-neutrophil interaction | - Cell-cell-interactions | - In vitro NET formation - Aggregate formation - Co-activation |
(−) sample clotting (−) no shear condition |
-Thrombus formation cascade - Activation -Extravasation / Migration / Bacterial bundling |
- Aggregate formation [125] - NET formation [126, 127] |
Abbreviations Table 1: ROS: Reactive oxygen species; NET: Neutrophil extracellular traps