Higher levels of adhesion markers ICAM-1 and VCAM-1 are induced on endothelial cells (ECs) activated by high-responder coculture supernatants vs. low-responder supernatants. Endothelial monolayers were treated for 24 hours with one of the following: conditioned coculture medium from a representative high-responder donor, a representative low-responder donor, or RPMI 1640/10% FCS as negative control medium. Cells were then dissociated and collected in a single-cell suspension using a nonprotease-based approach, and flow-cytometry analysis was used to monitor the surface expression levels of adhesion markers VCAM-1 and ICAM-1. Results for VCAM-1 levels are shown in the right panels, where in the resting state 0.7% of the cells were positive for VCAM-1 (black line), and the isotype (Iso) control is indicated by a filled gray curve area that very closely follows the resting-state cell levels In endothelial cells maintained in low-responder conditioned medium, 4% were positive for VCAM-1 (line), and in those maintained in high-responder conditioned medium, 77% were positive (line). Results for ICAM-1 levels in the same sample are shown in the left panels, where 66% of resting-state endothelial cells were positive for ICAM-1 at a relatively low level (line), and 73% of the cells treated with low-responder conditioned medium were ICAM-1 positive (line). These ICAM-1 levels were slightly higher than those in the resting state. A total of 96% of the cells were ICAM-1 positive in cells treated with the high-responder conditioned medium (line), and many of these cells were expressing high levels of ICAM-1. Relative fluorescent intensity is represented on the x-axis, and the cell numbers as counts are shown on the y-axis.