title: Multiparametric luminescence method for quantitative cell surface protein expression analysis and imaging.
abstract: A luminometric method for quantitative cell surface protein expression analysis has been developed in a microtiter plate format. The method is based on immunocytochemistry, the use of long-lived europium(III) and terbium(III) chelates and platinum(II) porphyrin luminescence labels in addition to short-lived syto13 DNA stain, and detection of photoluminescence emission from adhered cells by both time-resolved luminescence and conventional fluorescence. After the immunoreactions, the wells were evaporated to dryness, allowing repeated and postponed luminescence analysis even after months and cellular protein localization studies by microscopy imaging. The multiparametric method assayed the cell surface expression of ß1-integrin, E-selectin and intercellular adhesion molecule 1 (ICAM-1) in HUVE cells (human umbilical vein endothelial cells). The expression of E-selectin and ICAM-1 was enhanced by treating HUVECs with tumor necrosis factor α (TNF-α), while the expression level of ß1-integrin remained unchanged. The sensitivity limit of TNF-α detection by the method was ca. 1 pg/ml and the Z'-factors for the quantification of E-selectin and ICAM-1 were >0.7 suggesting a highly robust method. The novel approach proposed in this paper can be potentially applied to cell surface protein expression analysis in screening applications combined with localization studies of the target proteins by fluorescence microscopy imaging.
authors.affiliation: Laboratory of Biophysics, Department of Cell Biology and Anatomy and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, Turku, Finland. roohuttu@utu.fi
authors.forename: Roope
authors.initials: R
authors.lastname: Huttunen
authors.forename: Juhani
authors.initials: J
authors.lastname: Soini
authors.forename: Pirkko
authors.initials: P
authors.lastname: Härkönen
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D002478:Cells, Cultured; D019040:E-Selectin / Q000032:analysis; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D008163:Luminescent Measurements / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D019040:E-Selectin; D019012:Integrin beta1; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1