Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Abstract

Flow cytometric energy transfer (FCET)measurements between labeled specific sites of cell surface elements (Szollosi et al., Cytometry, 5210-216, 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 enabling 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.Key terms: Resonance energy transfer, concanavalin A, Gross virus leukemia, flow cytometry, flow microfluorimetry, receptor densityThe cytoplasmic cell surface membrane plays a major role in communication between the environment and the cell interior. The cell senses specific external stimuli through surface receptors. The information necessary for cell function can be communicated by the aggregation state of these receptors or by changes in the distribution state of receptors on the membrane.The proximity relationship of membrane receptors can be studied by measuring the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores conjugated to components in question (21). FRET measurements have been performed on cell surfaces using spectrofluorimetry (4,lB-201, microscopy (91, and flow cytometry (3,5,10,(13)(14)(15)24,25). The FRET efficiencies determined using spectrofluorimeters are values averaged over the entire homogeneous or heterogeneous population. The data obtained with spectrofluorimeters are highly perturbed by the amount of debris, dead cells, aggregates, etc. Furthermore the procedures used to eliminate excess labeled ligands also remove cells from the samples and present a n almost insurmountable problem when comparing fluorescence intensities of single-labeled samples with those of double-labeled samples. We have developed and recently published (5,24,25) a new method using a flow cytometer capable of dual-wavelength excitation to determine FRE...

Clustering of Class I HLA Oligomers with CD8 and TCR: Three-Dimensional Models Based on Fluorescence Resonance Energy Transfer and Crystallographic Data

“…In the present study, the FRET efficiency was determined in a Becton Dickinson (Sunnyvale, CA) FACStar Plus flow cytometer, by measuring both the extent of donor-quenching and acceptor sensitization through detecting spectrally selected fluorescence intensities on cell by cell basis, as described in details earlier (25,26,28). The efficiency of energy transfer (E) is expressed as percentage of the donor's (SFX) excitation energy tunneled to the acceptor (TAMRA-X) molecules.…”

Early endosome membrane dynamics characterized by flow cytometry

“…Flow cytometric FRET measurements have been applied in a simplified form using a flow cytometer equipped with a single excitation beam (29). The FACS 440 flow cytometer (Becton Dickinson, San Jose, CA) was used to determine energy transfer between FITC-BSA and TRITC-BSA present in EE populations.…”

“…Experimental values for S 1 ϭ 0.35 with 488 nm excitation and S 1 ϭ 0.15 with 514 nm excitation, S 4 ϭ 0.017 with 488 nm excitation and S 4 ϭ 0.068 with 514 nm excitation. As described elsewhere (29), E can be expressed as in equation 6:…”

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry

“…we employed fluorescence resonance energy transfer (FRET) combined with FCM (30). Assuming that ANT is an energy acceptor, we needed a fluorescent molecule presenting the following properties as donor: 1) permeant to living cells; 2) emission spectrum overlapping the excitation spectrum of ANT, and 3) good affinity binding to DNA to excite specifically the ANT intercalated in DNA.…”