Green fluorescent protein-based system for analysis of E-selectin-mediatedadhesion

Numerous cell-based or cell-free systems for study of selectin adhesion use radiolabeled tracers. However in addition to handling problems associated with the use of radioisotopes, these assays have difficulty relating a numb er of counts to a number of adherent cells. Here, we describe an assay that uses the natural fluorescence of the green fluorescent protein (GFP) to me asure binding of cells to E-selectin. We elaborated an adhesion system comp osed of a cell monolayer expressing E-selectin ligand to which monodisperse d fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are a dded. Due to GFP autofluorescence, adhered cells can be easily distinguishe d from cell monolayers by fluorescence microscopy, and adhesion can be meas ured by cytofluorometry. We applied this GFP-based adhesion assay to measur e the adherence of a pancreatic tumor cell line and found that the binding parameters of these cells satisfy a number of E-selectin-specific criteria.