Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single‐laser excitation at 458 nm

Abstract

Background: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser. Methods: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564 -606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in

TRAF3 Forms Heterotrimers with TRAF2 and Modulates Its Ability to Mediate NF-κB Activation

“…Plasmids-Plasmids that encode CFP-or YFP-fused to TRAF3 or -5 have been described previously (36). Where indicated, the fragments containing TRAF3 were cloned into the appropriate sites of HcRed-C1 to prepare HcRed-TRAF3.…”

“…The same PCR fragments for all TRAFs were cloned into His-tagged pcDNA3 (Invitrogen) to prepare His-TRAF2, His-TRAF3, His-TRAF5, and His-TRAF6 plasmids. A plasmid that encodes a FRETnegative control (CFP-TRAF2TRAF-YFP) has been previously described (36). All constructs were confirmed by DNA sequencing.…”

“…; BD Biosciences). The optical configuration for FRET measurement (FRET1) between CFP and YFP has been described previously (36,38). Briefly, the argon ion 488-nm laser line at 150 milliwatts and the krypton ion UV 407-nm laser line at 50 milliwatts were employed to excite the YFP and CFP, respectively.…”

Monitoring Caspase Activity in Living Cells Using Fluorescent Proteins and Flow Cytometry

“…The CFP-YFP FRET-positive control plasmid was described previously. 21,30 A plasmid with D-ϾA mutations (LEVD-ϾLEVA) in the caspase cleavage sites of the spacer of the CFP-LEVD-YFP plasmid, designated as CFP-14m-YFP, was made by inserting the annealed oligos 5Ј CC GGA GCA CTG GAG GTC GCA GCC CTG GAG GTC GCA C 3Ј and 5Ј CC GGG TGC GAC CTC CAG GGC TGC GAC CTC CAG TGC T 3Ј directly into the BspE I site of the CFP-YFP FRET-positive control. The human caspase 8 cDNA inserted into the pEGFP-N1 vector was described previously.…”

“…Recently we have developed an in vivo flow cytometric FRET method for measuring the molecular distance between CFP and YFP flurochromes. 21 Importantly, flow cytometry measures the FRET signals in a highthroughput manner and analyzes viable cells without any subjective selection of cells of interest. Additionally, when examined by flow cytometry, FRET-positive cells can be subsequently sorted to perform additional experiments.…”