Table 1:

Common In Vitro and Ex Vivo Models of Neutrophil Function

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