Parallel plate flow chamber assay to assess E-selectin ligand activity

To analyze the capacity of α(1,3)-fucosylated-hMSCs to engage vascular E-selectin under physiologic blood flow conditions, dynamic flow adhesion assays were performed using a parallel-plate flow chamber and binding interactions were visualized in real time using video-microscopy. All flow chamber studies were performed according to protocols previously described [11]. hMSCs were suspended in HBSS containing 10mM HEPES, 2mM CaCl₂ and 5% FBS and perfused through HUVEC-containing channels at an initial shear rate of 0.5 dynes/cm², with step-wise increments to 8 dynes/cm², thereby mimicking hemodynamic shear conditions present in the interaction of a blood-borne cell with human vasculature [16]. The total number of interacting cells in a single 4X field of view (fixed in the middle of the flow channel) as well as velocities and shear resistance during the 5 min perfusion period were evaluated to determine the capacity of cells to interact with endothelial E-selectin [17,18]. Controls for specificity of E-selectin binding consisted of perfusion of hMSC in presence of function-blocking anti-mouse E-selectin mAb (clone 10E9.6, from BD Biosciences).