Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Wiley - Tập 8 Số 2 - Trang 120-128 - 1987
János Szöllősi1, László Mátyus1, Lajos Trón1, Margit Balázs1, István Ember2, Mack J. Fulwyler3, Sándor Damjanovich1
1Departments of Biophysics, Medical University School, H‐4012 Debrecen, Hungary
2Departments of Hygiene and Epidemiology, Medical University School, H‐4012 Debrecen, Hungary
3Department of Laboratory Medicine, University of California, San Francisco, California 94143

Tóm tắt

AbstractFlow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Szöllosi et al., Cytometry, 5:210–216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: (1) The labeled ligands can be applied in excess, without washing, thereby enahling the investigation of relatively labile receptor‐ligand complexes. (2) Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. (3) The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. (4) In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell‐by‐cell basis, offering data on intramolecular conformational changes.This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.

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Wiley

Tập 8 Số 2

120-128

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