Isolation of FRET-positive cells using single 408-nm laser flow cytometry.
BACKGROUND
Flow cytometry may be used to isolate large amounts of living, fluorescently labeled cells. Certain fluorescent labels, like enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), allow the assessment of direct protein-protein interaction in situ, by fluorescence resonance energy transfer (FRET). However, current flow cytometric methods either require elaborate technical adaptations or, using a single laser protocol, are hampered by background signal. We optimized a single 408-nm laser protocol to detect FRET between ECFP/EYFP-tagged proteins.


METHODS
Cell lines stably expressing ECFP and/or EYFP or an EYFP-ECFP fusion protein were used to design the settings for the flow cytometer to detect FRET-positive cells using a single 408-nm laser. Using these settings, interactions between the subunits of the transcription factor NF-Y were studied.


RESULTS
Flow cytometric analysis of the cells expressing an EYFP-ECFP fusion protein yielded a discrete FRET-positive population. Using the same settings, in cells expressing NF-YB-CFP and NF-YC-YFP fusion proteins, FRET could also be detected. These cells were sorted and FRET was confirmed by confocal microscopy.


CONCLUSIONS
FRET-positive cells, expressing ECFP- and EYFP-tagged proteins, can be detected using single 408-nm laser excitation, with low background signal. This allows high-throughput analysis and isolation of viable FRET-positive and -negative cells for subsequent biological experiments.
van Wageningen
Sake
S
Central Hematology Laboratory of the University Medical Centre, St. Radboud, Geert Grooteplein zuid 8, 6500 HB Nijmegen, The Netherlands.
Pennings
Arie H
AH
van der Reijden
Bert A
BA
Boezeman
Jan B
JB
de Lange
Frank
F
Jansen
Joop H
JH
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins