A method for surface E-selectin site density determination

Abstract

E-selectin-mediated leukocyte adhesion on endothelial cells is a critical event in the inflammatory response. Site density determination of E-selectin on the substrate surface is the primary step in understanding the mechanism of E-selectin-induced cell adhesion in a parallel plate flow chamber. So far, the fluorescent method cannot measure low molecular density surfaces because of its weak sensitivity, and the traditional 125I radioiodination method can only detect the radioactivity of a solution in a tube. In this study, to measure low site density surfaces, a new method was constructed by combining 125I radioiodination with a GE Infinia Hawkeye 4 ECT. A saturation curve was obtained for the relationship between site densities and incubated concentrations of E-selectin, and a linear correlation was found within the range of low site density. Site densities were 9 and 140 sites/µm2 when polystyrene surfaces were incubated with E-selectin concentrations of 10 and 40 ng/ml, respectively. Under these densities, HL-60 cells could tether and roll on the substrates of the flow chamber. The radioiodination method developed in this study is an ideal detection method for even low-density surfaces because of its high sensitivity, which will provide better understanding of cell adhesion using a flow chamber.