Overview

The ability to monitor dynamic cell adhesion in vitro greatly contributes to our understanding of molecular mechanisms for recruitment of leukocytes on the sites of inflammation and other abnormal interactions between erythrocytes and vascular endothelial cells (ECs), such as vaso-occlusive crises in sickle cell anemia.

We have developed a novel flow-based cell adhesion assay that can characterize the multiple step adhesion process governed by the interactions between endothelial selectins (P- and E-selectin) and their counter ligands. With our proprietary approach we can assess the therapeutic effects of anti-adhesion agents in attenuation of cellular adhesive responses in whole blood on a range of pro-adhesive substrates.

Methods

Blood samples (whole or isolated cellular components) at baseline or following drug treatment (single dose or dose response) are subjected to physiologic flow across an adhesive substrate of interest (usually selectins). Time-lapse images are acquired to measure cell rolling / sliding along the channel surface. Mean velocity for rolling objects and cell flux are measured to generate a dynamic adhesion index (dAI).

Data Analysis

Data Analysis

The figure above shows a representative photomicrograph (left) and pseudo-color image of sickle leukocytes (right). We identify and track rolling cells with specialized software that allows us to select and represent cell movements by speed and vector. Leukocyte rolling on the pro-adhesive surface (e.g. P-selectin, E-Selectin, or activated endothelium layer) correlates to the degree of ligand-receptor interactions. Rolling cell density (cells/mm2)/rolling cell flux (cells/min), average rolling velocity (µm/s), and rolling cell percentage (%) are key parameters that can be easily obtained in our assay to characterize cell rolling behaviors and determine inflammatory states.

In these results the test compound did not significantly affect dynamic adhesion to p-selectin. Microfluidic channels were coated with p-selectin. Isolated leukocytes pretreated with vehicle or test compound was perfused through microchannels. Test compound (10mg/mL) did not significantly affect cell-rolling flux in dynamic adhesion assays to P-selectin (p = 0.51 and 0.94, isolated leukocytes and whole blood – data not shown, respectively)