Green fluorescent protein-based system for analysis of E-selectin-mediated adhesion.
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.
el-Battari
A
A
INSERM U 260, Faculté de Médecine, Marseille, France.
Zerfaoui
M
M
Panicot
L
L
Mas
E
E
Prévôt
C
C
Lombardo
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002450:Cell Communication / Q000187:drug effects / Q000235:genetics; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D019040:E-Selectin / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D004533:Egtazic Acid / Q000494:pharmacology; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D019040:E-Selectin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid