*Purpose: The Bioflux system has been used to study platelet-driven thrombosis and leukocyte adhesion. Typically, whole blood is stained with calcein (green) to evaluate platelet adhesion and aggregation but does not distinguish adherent leukocytes from larger platelet aggregates. We sought to develop a method to separately evaluate leukocyte (WBC) adhesion, which also take up calcein when whole blood is stained, while simultaneously measuring platelet aggregation during interactions between porcine endothelium and whole human blood.

*Methods: Porcine endothelia were cultured to confluence in microfluidic channels and then perfused with fluorescently stained human blood. Blood was separated into platelet-rich plasma (PRP), WBCs, and erythrocytes. In one condition, the 3 tubes were immediately recombined to reconstitute whole blood and then stained with calcein for 30 minutes (standard method). In the revised method, the PRP was stained with calcein and the WBCs were stained with Vybrant DiD for 30 minutes before recombining with erythrocytes as whole blood. The blood was then perfused at 5 dynes/cm² and 37^oC for one hour to mimic microcirculatory flow. Video images generated with the Montage software (Fluxion Biosciences) were overlaid to provide a visual representation of the staining.

*Results: Whole blood stained with calcein demonstrates heterogeneous green staining with a fine-grained speckled pattern (presumably reflecting individual adherent platelets) and larger particles reflecting adherent WBCs or platelet aggregates (Fig. 1A). When WBCs are separately labeled with DiD (red), the calcein-labeled platelet aggregates are readily distinguished from the adherent WBCs (Fig. 1B). These separate cell staining techniques are supported by preliminary flow cytometry analyses.

*Conclusions: Use of different fluorescent stains to separately identify platelets and leukocytes adds information useful to study the cell adhesion mechanisms associated with cross-species coagulation pathway dysregulation (physiologically inappropriate thrombosis, platelet activation and adhesion) and integrin- and selectin-mediated WBC activation and adhesive interactions. This technique can be applied to efficiently evaluate potentially important interactions between these cell populations that may contribute to graft injury mechanisms using a small sample of donor endothelial cells and a few milliliters of recipient blood.

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