Green Fluorescent Protein-Based System for Analysis of E-Selectin-Mediated Adhesion

Abstract

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 number 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 measure binding of cells to E-selectin. We elaborated an adhesion system composed of a cell monolayer expressing E-selectin ligand to which monodispersed fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are added. Due to GFP autofluorescence, adhered cells can be easily distinguished from cell monolayers by fluorescence microscopy, and adhesion can be measured by cytofluorometry. We applied this GFP-based adhesion assay to measure 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.

α(1,2)‐Fucosylation prevents sialyl Lewis x expression and E‐selectin‐mediated adhesion of fucosyltransferase VII‐transfected cells

“…It has been reported by Zöllner and Vestweber [38] that transfection of CHO cells with cDNA for FucT‐VII selectively activated the hamster homologue of the E‐selectin ligand‐1 (ESL‐1) in CHO cells. Similarly, we recently showed that FucT‐VII‐transfected CHO cells adhered well to E‐selectin [31]. Therefore, it was not necessary to transfect the ESL‐1 cDNA in our CHO cell lines to measure E‐selectin adhesion after FucT‐VII transfection.…”

“…In a reciprocal experiment, we investigated the possibility that HT transfection of CHO/FucT‐VII cells could suppress sLe x expression. In a first series of experiments, a CHO/FucT‐VII clone that expressed 100% sLe x [31] was transiently transfected by pcDNA3/HT construct or pcDNA3 vector alone and checked for sLe x expression. We performed a double‐labelling experiment in order to check whether cells which transiently express H epitope still express sLe x structures.…”

“…CHO/neo, CHO/ST6 and CHO/HT cells, transiently transfected with cDNA encoding FucT‐VII were assayed for adhesion to CHO cells expressing E‐selectin and EGFP (CHOgfp/E) as described previously [31]. Briefly, CHOgfp/E cells were harvested, washed twice in RPMI medium, resuspended at 0.5 × 10 6 cells·mL −1 in RPMI medium containing 1 mg·mL −1 BSA.…”