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

Abstract

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.Key terms: Lectins, receptor, flow analysis, flow cytometryThe state of receptor aggregation in the cytoplasmic membrane may play a central role in the mediation between external stimuli and the complex metabolic processes of the cell. For example, binding of epidermal growth factor (111, nerve growth factor (91, cr2-macroglobulin (lo), and insulin (12) to their corresponding receptors results in a redistribution of the latter followed by a series of prompt biological responses, and, in the case of the hormones, initiation of DNA synthesis and stimulation of growth. The importance of receptor redistribution for normal metabolism has been investigated with mutant cells. Anderson et a1 (1) demonstrated that in one genetic form of the metabolic disorder hypercholesterolemia the low-density-lipoprotein receptors can no longer cluster, a situation that prevents the internalisation of the protein receptor complex. Specific cell types or different functional surfaces of differentiated cells may exhibit distinct spatial distributions of surface proteins, eg, as in the case of Con A binding glycoproteins on endothelial cells (13). The role of such microdomains in cell-cell recognition or functional compartmentalization is the subject of many current investigations.One of the most useful techniques for studying proximity relationships (intermolecular distances) in the range that pertains to the cell membrane is fluorescence resonance energy transfer. The method has been applied to the study of cell surface phenomena using steadystate fluorimeters (51, fluorescence microscopes (6), and flow cytometers (4,18,14). We have recently developed a method for the quantitative evaluation of the energy transfer efficiency (E) between donors and acceptors on cell...

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins

“…Donor and acceptor emissions were separated by a 525-nm dichroic mirror and were detected through 487/37-nm and 560/70-nm bandpass mirrors, respectively. Donor, acceptor, and FRET intensities were recorded as described previously (17,18). Briefly, two fluorescence intensities were detected for the donor excitation at 457 nm (donor channel I 1 , detection at 487 nm; acceptor channel I 2 , emission corresponding to the FRET channel at 560 nm), and one intensity was measured for the acceptor excitation at 514 nm (I 3 , acceptor emission at 560 nm).…”

“…Therefore, three independent measurements have to be taken that correspond to the donor (I 1 ), FRET (I 2 ), and acceptor (I 3 ) channels (17,18):…”

“…S 1 corrects for the overspill of donor fluorescence from I 1 to I 2 , S 3 for the overspill of donor fluorescence from I 1 to I 3 , and S 2 for the overspill of acceptor emission from I 3 to I 2 . The constants S 1 to S 3 are determined on single-labeled samples as described previously (17,18) and can be expressed by the following equations:…”

“…We previously reported one such method based on the combined detection of donor and acceptor fluorescence intensities in cells labeled with fluorescent ligands or antibodies using flow cytometry (17,18). Later, we established a similar method in fluorescence microscopy (19).…”

“…These methods require a factor, variably called a (17)(18)(19) or G (11,13), which relates the intensity lost on the donor side due to donor quenching to the enhanced emission of the acceptor due to sensitized emission. Methods for the determination of a have been reported for antibody-labeled cells in flow cytometry (17,18) and microscopy (19) and lately for VFP-expressing cells in microscopy (13). To the best of our knowledge, successful determination of a has not been reported for VFP-expressing cells using flow cytometry.…”