Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady-state fluorimetric and flow cytometric methods

Cytometry. 1984 Mar;5(2):210-6. doi: 10.1002/cyto.990050216.


The energy transfer efficiency E was measured between fluorescein-conjugated concanavalin A (Con A) and rhodamine-conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady-state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady-state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor-ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light-scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor-acceptor redistribution on subpopulations can be measured.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cell Membrane / physiology*
  • Energy Transfer
  • Flow Cytometry / methods
  • Lymphoma / pathology
  • Lymphoma / physiopathology*
  • Mathematics
  • Mice
  • Mice, Inbred Strains
  • Microscopy, Fluorescence / methods