Analysis insights for three FRET pairs of chemically unlinked two‐molecule FRET cytometry

Förster resonance energy transfer (FRET) is the direct energy exchange between two‐component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein–protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor‐1 (TNFR1) molecules expressed as recombinant C‐terminal fusion proteins of cyan, yellow, or red fluorescent protein (‐CFP, ‐YFP, or ‐RFP) to evaluate two‐molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET‐1), YFP→RFP (FRET‐2) and CFP→RFP (FRET‐3), by comparing TNFR1+TNFR1 with non‐interacting TNFR1+CD27 proteins, on both LSR‐II and Fortessa X‐20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of “spiked” FRET donor cells, together with TNFR1+TNFR1 co‐transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single‐cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single‐cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1‐based system the mean CFP→YFP FRET‐1 efficiency is 0.43 (LSR‐II) and 0.41 (Fortessa X‐20), the mean YFP→RFP FRET‐2 efficiency is 0.30 (LSR‐II) and 0.29 (Fortessa X‐20), and the mean CFP→RFP FRET‐3 efficiency is 0.56 (LSR‐II) and 0.54 (Fortessa X‐20). This study also embraces multi‐dimensional clustering using t‐SNE, Fit‐SNE, UMAP, Tri‐Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.