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Related Experiment Video

Updated: Jun 4, 2026

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
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Multiparametric luminescence method for quantitative cell surface protein expression analysis and imaging.

Roope Huttunen1, Juhani Soini, Pirkko Härkönen

  • 1Laboratory of Biophysics, Department of Cell Biology and Anatomy and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, Turku, Finland. roohuttu@utu.fi

Journal of Immunological Methods
|February 1, 2011
PubMed
Summary

A new luminometric method quantifies cell surface protein expression using advanced luminescence labels. This robust technique can analyze protein levels and localization, aiding drug discovery and diagnostics.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Analytical Chemistry

Background:

  • Quantitative analysis of cell surface proteins is crucial for understanding cellular functions and disease mechanisms.
  • Existing methods for cell surface protein expression analysis often lack sensitivity, robustness, or the ability to perform localization studies.
  • Developing novel, high-throughput methods for cell surface protein analysis is essential for drug screening and biomarker discovery.

Purpose of the Study:

  • To develop and validate a novel luminometric method for quantitative cell surface protein expression analysis in a microtiter plate format.
  • To assess the method's ability to perform multiparametric analysis and cellular protein localization studies.
  • To evaluate the method's sensitivity and robustness for detecting changes in protein expression.

Main Methods:

  • Development of a luminometric assay utilizing immunocytochemistry with long-lived europium(III), terbium(III), and platinum(II) porphyrin luminescence labels.
  • Detection of photoluminescence from adhered cells using time-resolved luminescence and conventional fluorescence after sample evaporation for postponed analysis.
  • Assay of cell surface protein expression (ß1-integrin, E-selectin, ICAM-1) in human umbilical vein endothelial cells (HUVECs) and assessment of tumor necrosis factor α (TNF-α) effects.

Main Results:

  • The method successfully quantified cell surface protein expression of ß1-integrin, E-selectin, and ICAM-1 in HUVECs.
  • Treatment with TNF-α significantly enhanced E-selectin and ICAM-1 expression, while ß1-integrin levels remained unchanged.
  • The method demonstrated high sensitivity for TNF-α detection (ca. 1 pg/ml) and robust performance with Z'-factors >0.7 for E-selectin and ICAM-1 quantification.
  • The assay allowed for postponed luminescence analysis and cellular protein localization studies via microscopy imaging.

Conclusions:

  • A novel, robust, and sensitive luminometric method for quantitative cell surface protein expression analysis has been established.
  • The developed method enables multiparametric analysis, postponed measurements, and simultaneous protein localization studies.
  • This approach holds significant potential for high-throughput screening applications in drug discovery and diagnostics.