Encyclopedia of Experiments: Immunology
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Transcript
Take pre-assembled flow chambers connected to flow systems with transwell filters: One with an endothelial monoculture on top and the other with an endothelial culture on top and stromal cells on the bottom.
Both chambers are pre-treated with a pro-inflammatory cytokine.
In a monoculture, cytokines activate endothelial cells, upregulating adhesion molecule expression.
However, cross-talk between stromal and endothelial cells in the co-culture leads to stromal cells producing regulatory mediators that downregulate the endothelial cells' response to pro-inflammatory cytokines.
Position the chamber on an inverted phase-contrast microscope stage.
Introduce a neutrophil suspension.
Neutrophils get recruited and bind to the endothelial adhesion molecules via transient adhesive interactions, facilitating their interaction with surface-bound chemokines, and rolling along the endothelium.
This interaction activates the integrins on neutrophils, stabilizing the adhesion and resulting in transmigration across the endothelial cells.
However, in the co-culture, the downregulated endothelial response results in suppressed neutrophil recruitment.
Visualize the cultures under phase-contrast microscopy to assess neutrophil recruitment from the flow.
