Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins
Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is no...
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| Veröffentlicht in: | Cytometry. Part A 2014-07, Vol.85 (7), p.621-627 |
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| container_title | Cytometry. Part A |
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| creator | Heinen, André P. Wanke, Florian Moos, Sonja Attig, Sebastian Luche, Hervé Pal, Prajna Paramita Budisa, Nediljko Fehling, Hans Jörg Waisman, Ari Kurschus, Florian C. |
| description | Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. © 2014 International Society for Advancement of Cytometry |
| doi_str_mv | 10.1002/cyto.a.22451 |
| format | Article |
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In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. © 2014 International Society for Advancement of Cytometry</description><identifier>ISSN: 1552-4922</identifier><identifier>EISSN: 1552-4930</identifier><identifier>DOI: 10.1002/cyto.a.22451</identifier><identifier>PMID: 24616430</identifier><language>eng</language><publisher>United States</publisher><subject>Animals ; Cytokines - analysis ; Cytoplasm - metabolism ; EGFP ; EYFP ; fate mapping ; fixation ; Fixatives ; flow cytometry ; Flow Cytometry - methods ; Fluorescent Dyes ; Forkhead Transcription Factors ; formaldehyde ; FoxP3 ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; mouse T cells ; Mycobacterium tuberculosis - immunology ; Nuclear Proteins - analysis ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; RFP ; RORγt ; Staining and Labeling ; T-Box Domain Proteins ; T-Lymphocytes - cytology ; Tissue Fixation - methods ; Transcription Factors - analysis ; T‐bet</subject><ispartof>Cytometry. 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Part A</title><addtitle>Cytometry A</addtitle><description>Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. © 2014 International Society for Advancement of Cytometry</description><subject>Animals</subject><subject>Cytokines - analysis</subject><subject>Cytoplasm - metabolism</subject><subject>EGFP</subject><subject>EYFP</subject><subject>fate mapping</subject><subject>fixation</subject><subject>Fixatives</subject><subject>flow cytometry</subject><subject>Flow Cytometry - methods</subject><subject>Fluorescent Dyes</subject><subject>Forkhead Transcription Factors</subject><subject>formaldehyde</subject><subject>FoxP3</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Mice, Transgenic</subject><subject>mouse T cells</subject><subject>Mycobacterium tuberculosis - immunology</subject><subject>Nuclear Proteins - analysis</subject><subject>Nuclear Receptor Subfamily 1, Group F, Member 3</subject><subject>RFP</subject><subject>RORγt</subject><subject>Staining and Labeling</subject><subject>T-Box Domain Proteins</subject><subject>T-Lymphocytes - cytology</subject><subject>Tissue Fixation - methods</subject><subject>Transcription Factors - analysis</subject><subject>T‐bet</subject><issn>1552-4922</issn><issn>1552-4930</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc9PwyAUx4nRuDm9eTYcPdgJFNr1uCz-SpbsMg-eCKUPV9OWCa3L_nupnTsaTxDehw-870PompIpJYTd631rp2rKGBf0BI2pECziWUxOj3vGRujC-w9CYkFido5GjCc04TEZI3ipt85-QYFraDe2wK3FDlpVNrgXe1uVOhxsrWvB4YC2EEqm6qwDr6HRgHebsgLs-ztl846NdbjpdAXqyPtLdGZU5eHqsE7Q6-PDevEcLVdPL4v5MtKCCBplWusZS7kShdaGc8NIzjPOU6ZSShIDPM0zI0SWFIQpBiwpYkrpTOnQTi7yeIJuB294-LMD38q6DL-sKtWA7bykgsdZlqap-A9KKGNkNgvo3YBqZ713YOTWlbVye0mJ7Gcg-6ikkj8zCPjNwdzlNRRH-Df0APAB2IXg9n_K5OJtvZoP3m9ZwJSa</recordid><startdate>201407</startdate><enddate>201407</enddate><creator>Heinen, André P.</creator><creator>Wanke, Florian</creator><creator>Moos, Sonja</creator><creator>Attig, Sebastian</creator><creator>Luche, Hervé</creator><creator>Pal, Prajna Paramita</creator><creator>Budisa, Nediljko</creator><creator>Fehling, Hans Jörg</creator><creator>Waisman, Ari</creator><creator>Kurschus, Florian C.</creator><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><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>201407</creationdate><title>Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins</title><author>Heinen, André P. ; Wanke, Florian ; Moos, Sonja ; Attig, Sebastian ; Luche, Hervé ; Pal, Prajna Paramita ; Budisa, Nediljko ; Fehling, Hans Jörg ; Waisman, Ari ; Kurschus, Florian C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5051-9ccc8274a5dccf44f20b494472a7106fe47b9f5596d02a2e26d31118ac616b5b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>Cytokines - analysis</topic><topic>Cytoplasm - metabolism</topic><topic>EGFP</topic><topic>EYFP</topic><topic>fate mapping</topic><topic>fixation</topic><topic>Fixatives</topic><topic>flow cytometry</topic><topic>Flow Cytometry - methods</topic><topic>Fluorescent Dyes</topic><topic>Forkhead Transcription Factors</topic><topic>formaldehyde</topic><topic>FoxP3</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Mice, Transgenic</topic><topic>mouse T cells</topic><topic>Mycobacterium tuberculosis - immunology</topic><topic>Nuclear Proteins - analysis</topic><topic>Nuclear Receptor Subfamily 1, Group F, Member 3</topic><topic>RFP</topic><topic>RORγt</topic><topic>Staining and Labeling</topic><topic>T-Box Domain Proteins</topic><topic>T-Lymphocytes - cytology</topic><topic>Tissue Fixation - methods</topic><topic>Transcription Factors - analysis</topic><topic>T‐bet</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Heinen, André P.</creatorcontrib><creatorcontrib>Wanke, Florian</creatorcontrib><creatorcontrib>Moos, Sonja</creatorcontrib><creatorcontrib>Attig, Sebastian</creatorcontrib><creatorcontrib>Luche, Hervé</creatorcontrib><creatorcontrib>Pal, Prajna Paramita</creatorcontrib><creatorcontrib>Budisa, Nediljko</creatorcontrib><creatorcontrib>Fehling, Hans Jörg</creatorcontrib><creatorcontrib>Waisman, Ari</creatorcontrib><creatorcontrib>Kurschus, Florian C.</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><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Cytometry. 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In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells. © 2014 International Society for Advancement of Cytometry</abstract><cop>United States</cop><pmid>24616430</pmid><doi>10.1002/cyto.a.22451</doi><tpages>7</tpages></addata></record> |
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| source | MEDLINE; Access via Wiley Online Library; EZB-FREE-00999 freely available EZB journals; Wiley Online Library (Open Access Collection); Alma/SFX Local Collection |
| subjects | Animals Cytokines - analysis Cytoplasm - metabolism EGFP EYFP fate mapping fixation Fixatives flow cytometry Flow Cytometry - methods Fluorescent Dyes Forkhead Transcription Factors formaldehyde FoxP3 Mice Mice, Inbred C57BL Mice, Transgenic mouse T cells Mycobacterium tuberculosis - immunology Nuclear Proteins - analysis Nuclear Receptor Subfamily 1, Group F, Member 3 RFP RORγt Staining and Labeling T-Box Domain Proteins T-Lymphocytes - cytology Tissue Fixation - methods Transcription Factors - analysis T‐bet |
| title | Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins |
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