An In Vitro Flow Investigation of the Effect of Stromal Cells on Leukocyte Recruitment

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.