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.

Keywords:

• E-selectin
• site density determination
• ¹²⁵I radioiodination
• SPECT

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [grant 31200705] (LQH), [31170887] (WJH) and [11272125] (YF), and Fundamental Research Funds for the Central Universities [2014ZZ0051] (LQH).