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.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004735:Energy Transfer; D005434:Flow Cytometry / Q000379:methods; D008223:Lymphoma / Q000473:pathology / Q000503:physiopathology*; D008433:Mathematics; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence / Q000379:methods