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

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.