FRET in the Analysis of In Vitro Cell–Cell Fusion by Flow Cytometry

Abstract

Cell-cell fusion is a frequent event in nature leading to modification of cell fate. In this chapter, we describe a flow cytometric procedure for the quantitative assessment of in vitro cell-cell fusion events that allows the discrimination of fused from aggregated cells. The assay is based on the differential labeling of fusion partners with lipophilic fluorescent probes DiI (red) and DiO (green). Double fluorescent fused cells can be detected after coculturing by means of a flow cytometer equipped with a 488 nm laser. Fusion events can be distinguished from cell aggregates by the enhancement of the DiI red fluorescence intensity due to resonance energy transfer between the two probes occurring in the fused but not in the aggregated cell population.

Mesenchymal stem cell‐glioblastoma interactions mediated via kinin receptors unveiled by cytometry

“…Exchange of cell membranes, vesicles or cell fusion can be detected and quantified by image overlay or flow cytometry (by gating of, i.e., dsRed‐(U87) fluorescence versus Dio‐(MSC) fluorescence (Figure 2(A) and (C); further details are provided in Appendix S1 (MIFlowCyt) or eGFP (U373) versus Dil‐(MSC) fluorescence (Figure 2(B)). Image and flow cytometry measurements of vesicle transfer/cell fusion can also be determined with a Fluorescence Resonance Energy Transfer (FRET) method using Dil and Dio probes [112]. Flow cytometry measurement at 488 nm excites DiO fluorescence, while only Dil fluorescence at this wavelength is poorly excited.…”