Transmigration of adherent human monocytes decorated with platelets under flow. (A) Human monocytes decorated with platelets (NycoPrep preparation) were captured onto HUVEC luminal surfaces under flow (separate panels; black stars). The attached platelets switched adhesion from monocyte to endothelial luminal surfaces (white arrows) as the monocyte underwent transmigration into the abluminal compartment (white stars). The composite image demonstrates that the platelets remained adherent under flow at the original site of transmigration on the endothelium (red area) during the course of monocyte abluminal migration (2-8 minutes; green area). (B) Monocytes purified by NycoPrep displayed high levels of the platelet marker CD41 and of JAM-C. Monocytes (CD14⁺) purified using NycoPrep displayed higher, more heterogeneous levels of CD41 (top right quadrant; 28% total monocyte population). Histogram of JAM-C expression on gated CD14⁺ cells also showed high heterogeneous levels of JAM-C on membrane surfaces (▩). (C) Monocytes purified by negative selection displayed markers associated with adherent platelets. Monocytes (CD14⁺) purified by negative selection and devoid of visible platelets still displayed lower levels of CD41 (CD41⁺ population designated in top right quadrant; 73% total monocyte population). Histogram of JAM-C expression confirmed expression of JAM-C but at lower levels (▩) to monocytes prepared by NycoPrep. (D) Monocyte-associated JAM-C expression was lost after transmigration. Transmigrated monocytes (95%+) in HUVEC coculture under flow were rescued by trypsinisation and labeled with an antibody to JAM-C. Analysis by flow cytometry of monocyte suspensions treated with trypsin remained JAM-C⁺ (▩; left panel), but rescued monocytes from trypsinized HUVEC–monocyte suspensions were JAM-C⁻ (▩; right panel).