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 |
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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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fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_67807687</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>67807687</sourcerecordid><originalsourceid>FETCH-LOGICAL-c3334-15e4c1af234b77c78f9f1ca6dea27ad05501a31550d1fd712901409a40ef03583</originalsourceid><addsrcrecordid>eNp9kL1OwzAURi0EolDYmJEnJlKufxInI6paqFRUCZWByXITGwU5cYkTqmw8As_Ik5CQCjaWe-9wdPTdD6ELAhMCQG_StnYTNaFAQ36ATkgY0oAnDA5_b0pH6NT7VwAWAqPHaEQinsRRHJ2gh4V3VtW5K7EzeP44W399fG6dz-v8XeNUW-tx4_PyBffDaswh7oiywFZ5XWFj3Q73EQpdV-0ZOjLKen2-32P0NJ-tp_fBcnW3mN4ug5QxxgMSap4SZSjjGyFSEZvEkFRFmVZUqAzCEIhiXXrIiMkEoQkQDonioE33Q8zG6Grwbiv31mhfyyL3fVhVatd4GYkYRBSLDrwewLRy3lfayG2VF6pqJQHZ1yf77FLJn_o6_HLvbTaFzv7gfV8dwAZgl1vd_iuT0-f1atB-A1L2fRU</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>67807687</pqid></control><display><type>article</type><title>Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry</title><source>MEDLINE</source><source>Wiley Online Library Free Content</source><source>Access via Wiley Online Library</source><source>EZB-FREE-00999 freely available EZB journals</source><source>Alma/SFX Local Collection</source><creator>van Wageningen, Sake ; Pennings, Arie H. ; van der Reijden, Bert A. ; Boezeman, Jan B. ; de Lange, Frank ; Jansen, Joop H.</creator><creatorcontrib>van Wageningen, Sake ; Pennings, Arie H. ; van der Reijden, Bert A. ; Boezeman, Jan B. ; de Lange, Frank ; Jansen, Joop H.</creatorcontrib><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</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. Part A, 2006-04, Vol.69A (4), p.291-298</ispartof><rights>Copyright © 2006 Wiley‐Liss, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3334-15e4c1af234b77c78f9f1ca6dea27ad05501a31550d1fd712901409a40ef03583</citedby><cites>FETCH-LOGICAL-c3334-15e4c1af234b77c78f9f1ca6dea27ad05501a31550d1fd712901409a40ef03583</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcyto.a.20254$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcyto.a.20254$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,1433,27924,27925,45574,45575,46409,46833</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16498686$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>van Wageningen, Sake</creatorcontrib><creatorcontrib>Pennings, Arie H.</creatorcontrib><creatorcontrib>van der Reijden, Bert A.</creatorcontrib><creatorcontrib>Boezeman, Jan B.</creatorcontrib><creatorcontrib>de Lange, Frank</creatorcontrib><creatorcontrib>Jansen, Joop H.</creatorcontrib><title>Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry</title><title>Cytometry. Part A</title><addtitle>Cytometry A</addtitle><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</description><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - metabolism</subject><subject>Cell Line, Tumor</subject><subject>Cell Separation - instrumentation</subject><subject>Cell Separation - methods</subject><subject>ECFP</subject><subject>EYFP</subject><subject>flow cytometry</subject><subject>Flow Cytometry - instrumentation</subject><subject>Flow Cytometry - methods</subject><subject>Fluorescence Resonance Energy Transfer - instrumentation</subject><subject>Fluorescence Resonance Energy Transfer - methods</subject><subject>FRET</subject><subject>Green Fluorescent Proteins - chemistry</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Humans</subject><subject>Luminescent Proteins - chemistry</subject><subject>Luminescent Proteins - metabolism</subject><subject>Microscopy, Confocal</subject><subject>NF‐Y</subject><issn>1552-4922</issn><issn>1552-4930</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kL1OwzAURi0EolDYmJEnJlKufxInI6paqFRUCZWByXITGwU5cYkTqmw8As_Ik5CQCjaWe-9wdPTdD6ELAhMCQG_StnYTNaFAQ36ATkgY0oAnDA5_b0pH6NT7VwAWAqPHaEQinsRRHJ2gh4V3VtW5K7EzeP44W399fG6dz-v8XeNUW-tx4_PyBffDaswh7oiywFZ5XWFj3Q73EQpdV-0ZOjLKen2-32P0NJ-tp_fBcnW3mN4ug5QxxgMSap4SZSjjGyFSEZvEkFRFmVZUqAzCEIhiXXrIiMkEoQkQDonioE33Q8zG6Grwbiv31mhfyyL3fVhVatd4GYkYRBSLDrwewLRy3lfayG2VF6pqJQHZ1yf77FLJn_o6_HLvbTaFzv7gfV8dwAZgl1vd_iuT0-f1atB-A1L2fRU</recordid><startdate>200604</startdate><enddate>200604</enddate><creator>van Wageningen, Sake</creator><creator>Pennings, Arie H.</creator><creator>van der Reijden, Bert A.</creator><creator>Boezeman, Jan B.</creator><creator>de Lange, Frank</creator><creator>Jansen, Joop H.</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>200604</creationdate><title>Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry</title><author>van Wageningen, Sake ; Pennings, Arie H. ; van der Reijden, Bert A. ; Boezeman, Jan B. ; de Lange, Frank ; Jansen, Joop H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3334-15e4c1af234b77c78f9f1ca6dea27ad05501a31550d1fd712901409a40ef03583</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - metabolism</topic><topic>Cell Line, Tumor</topic><topic>Cell Separation - instrumentation</topic><topic>Cell Separation - methods</topic><topic>ECFP</topic><topic>EYFP</topic><topic>flow cytometry</topic><topic>Flow Cytometry - instrumentation</topic><topic>Flow Cytometry - methods</topic><topic>Fluorescence Resonance Energy Transfer - instrumentation</topic><topic>Fluorescence Resonance Energy Transfer - methods</topic><topic>FRET</topic><topic>Green Fluorescent Proteins - chemistry</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Humans</topic><topic>Luminescent Proteins - chemistry</topic><topic>Luminescent Proteins - metabolism</topic><topic>Microscopy, Confocal</topic><topic>NF‐Y</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>van Wageningen, Sake</creatorcontrib><creatorcontrib>Pennings, Arie H.</creatorcontrib><creatorcontrib>van der Reijden, Bert A.</creatorcontrib><creatorcontrib>Boezeman, Jan B.</creatorcontrib><creatorcontrib>de Lange, Frank</creatorcontrib><creatorcontrib>Jansen, Joop H.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Cytometry. Part A</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>van Wageningen, Sake</au><au>Pennings, Arie H.</au><au>van der Reijden, Bert A.</au><au>Boezeman, Jan B.</au><au>de Lange, Frank</au><au>Jansen, Joop H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation of FRET‐positive cells using single 408‐nm laser flow cytometry</atitle><jtitle>Cytometry. Part A</jtitle><addtitle>Cytometry A</addtitle><date>2006-04</date><risdate>2006</risdate><volume>69A</volume><issue>4</issue><spage>291</spage><epage>298</epage><pages>291-298</pages><issn>1552-4922</issn><eissn>1552-4930</eissn><abstract>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</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>16498686</pmid><doi>10.1002/cyto.a.20254</doi><tpages>8</tpages></addata></record> |
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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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