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 flu...

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Veröffentlicht in:Cytometry. Part A 2006-04, Vol.69A (4), p.291-298
Hauptverfasser: van Wageningen, Sake, Pennings, Arie H., van der Reijden, Bert A., Boezeman, Jan B., de Lange, Frank, Jansen, Joop H.
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container_end_page 298
container_issue 4
container_start_page 291
container_title Cytometry. Part A
container_volume 69A
creator van Wageningen, Sake
Pennings, Arie H.
van der Reijden, Bert A.
Boezeman, Jan B.
de Lange, Frank
Jansen, Joop H.
description 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. Conclusion: 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. © 2006 International Society for Analytical Cytology
doi_str_mv 10.1002/cyto.a.20254
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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. Conclusion: 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. © 2006 International Society for Analytical Cytology</description><identifier>ISSN: 1552-4922</identifier><identifier>EISSN: 1552-4930</identifier><identifier>DOI: 10.1002/cyto.a.20254</identifier><identifier>PMID: 16498686</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Bacterial Proteins - chemistry ; Bacterial Proteins - metabolism ; Cell Line, Tumor ; Cell Separation - instrumentation ; Cell Separation - methods ; ECFP ; EYFP ; flow cytometry ; Flow Cytometry - instrumentation ; Flow Cytometry - methods ; Fluorescence Resonance Energy Transfer - instrumentation ; Fluorescence Resonance Energy Transfer - methods ; FRET ; Green Fluorescent Proteins - chemistry ; Green Fluorescent Proteins - metabolism ; Humans ; Luminescent Proteins - chemistry ; Luminescent Proteins - metabolism ; Microscopy, Confocal ; NF‐Y</subject><ispartof>Cytometry. 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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. Conclusion: FRET‐positive cells, expressing ECFP‐ and EYFP‐tagged proteins, can be detected using single 408‐nm laser excitation, with low background signal. 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subjects Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Cell Line, Tumor
Cell Separation - instrumentation
Cell Separation - methods
ECFP
EYFP
flow cytometry
Flow Cytometry - instrumentation
Flow Cytometry - methods
Fluorescence Resonance Energy Transfer - instrumentation
Fluorescence Resonance Energy Transfer - methods
FRET
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - metabolism
Humans
Luminescent Proteins - chemistry
Luminescent Proteins - metabolism
Microscopy, Confocal
NF‐Y
title Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry
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