Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands
Background Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor‐ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled lig...
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Veröffentlicht in: | Cytometry (New York, N.Y.) N.Y.), 2001-10, Vol.45 (2), p.102-114 |
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creator | Waller, Anna Pipkorn, David Sutton, Karyn L. Linderman, Jennifer J. Omann, Geneva M. |
description | Background
Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor‐ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N‐formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands.
Methods
Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO‐NLFNYK‐tetramethylrhodamine.
Results
Calibration parameters were determined for six fluorescently‐labeled N‐formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO‐NLFNYK‐tetramethylrhodamine determined directly or in competition with CHO‐NLFNYK‐fluorescein were statistically identical.
Conclusions
Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated. Cytometry 45:102–114, 2001. © 2001 Wiley‐Liss, Inc. |
doi_str_mv | 10.1002/1097-0320(20011001)45:2<102::AID-CYTO1152>3.0.CO;2-Z |
format | Article |
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Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor‐ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N‐formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands.
Methods
Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO‐NLFNYK‐tetramethylrhodamine.
Results
Calibration parameters were determined for six fluorescently‐labeled N‐formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO‐NLFNYK‐tetramethylrhodamine determined directly or in competition with CHO‐NLFNYK‐fluorescein were statistically identical.
Conclusions
Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated. Cytometry 45:102–114, 2001. © 2001 Wiley‐Liss, Inc.</description><identifier>ISSN: 0196-4763</identifier><identifier>EISSN: 1097-0320</identifier><identifier>DOI: 10.1002/1097-0320(20011001)45:2<102::AID-CYTO1152>3.0.CO;2-Z</identifier><identifier>PMID: 11590622</identifier><language>eng</language><publisher>New York: John Wiley & Sons, Inc</publisher><subject>binding kinetics ; Binding, Competitive ; Calibration ; flow cytometry ; Flow Cytometry - methods ; fluorescence calibration ; Fluorescent Dyes - chemistry ; Fluorescent Dyes - metabolism ; G‐protein coupled receptor ; Humans ; Ligands ; neutrophils ; Neutrophils - metabolism ; N‐formyl peptide receptor ; Oligopeptides - chemistry ; Oligopeptides - metabolism ; Protein Binding ; Reproducibility of Results ; Spectrometry, Fluorescence - methods</subject><ispartof>Cytometry (New York, N.Y.), 2001-10, Vol.45 (2), p.102-114</ispartof><rights>Copyright © 2001 Wiley‐Liss, Inc.</rights><rights>Copyright 2001 Wiley-Liss, Inc.</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c5092-2e1d2675242fa3c8a1c3f59ce01c64bd57c30db9c4a25b21c49dd3f09abec5db3</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%2F1097-0320%2820011001%2945%3A2%3C102%3A%3AAID-CYTO1152%3E3.0.CO%3B2-Z$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2F1097-0320%2820011001%2945%3A2%3C102%3A%3AAID-CYTO1152%3E3.0.CO%3B2-Z$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>315,781,785,1418,1434,27929,27930,45579,45580,46414,46838</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11590622$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Waller, Anna</creatorcontrib><creatorcontrib>Pipkorn, David</creatorcontrib><creatorcontrib>Sutton, Karyn L.</creatorcontrib><creatorcontrib>Linderman, Jennifer J.</creatorcontrib><creatorcontrib>Omann, Geneva M.</creatorcontrib><title>Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands</title><title>Cytometry (New York, N.Y.)</title><addtitle>Cytometry</addtitle><description>Background
Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor‐ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N‐formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands.
Methods
Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO‐NLFNYK‐tetramethylrhodamine.
Results
Calibration parameters were determined for six fluorescently‐labeled N‐formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO‐NLFNYK‐tetramethylrhodamine determined directly or in competition with CHO‐NLFNYK‐fluorescein were statistically identical.
Conclusions
Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated. Cytometry 45:102–114, 2001. © 2001 Wiley‐Liss, Inc.</description><subject>binding kinetics</subject><subject>Binding, Competitive</subject><subject>Calibration</subject><subject>flow cytometry</subject><subject>Flow Cytometry - methods</subject><subject>fluorescence calibration</subject><subject>Fluorescent Dyes - chemistry</subject><subject>Fluorescent Dyes - metabolism</subject><subject>G‐protein coupled receptor</subject><subject>Humans</subject><subject>Ligands</subject><subject>neutrophils</subject><subject>Neutrophils - metabolism</subject><subject>N‐formyl peptide receptor</subject><subject>Oligopeptides - chemistry</subject><subject>Oligopeptides - metabolism</subject><subject>Protein Binding</subject><subject>Reproducibility of Results</subject><subject>Spectrometry, Fluorescence - methods</subject><issn>0196-4763</issn><issn>1097-0320</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVUU1vEzEQtRCIhsJfQD4hOGwY2-vdOFRI1fJVqVIuAam9WF7bW4y869R2qMKvx1HSwoUDp5HevI_RPITOCMwJAH1LQLQVMAqvKQApEHlT8yUte7pcnl98qLqr9YoQTt-zOcy71TtaXT9CswfZYzQDIpqqbht2gp6l9AMARFOzp-ikyAQ0lM7Q7TflnVHZhQmHAQ8-3GG9y2G0OTqNdRg3Nrvsflrcu8m46QZvYshBB5-wmgzW31VUOtvofv3lsg3RJm2n7HfYq956a7B3N0WQnqMng_LJvjjOU_T108d196W6XH2-6M4vK81B0IpaYmjTclrTQTG9UESzgQttgeim7g1vNQPTC10ryntKdC2MYQOIkqa56dkpenXwLffebm3KcnTlJO_VZMM2yZaSdsEXUIjrA1HHkFK0g9xEN6q4kwTkvgq5_6nc_1TeVyFrLvc4lbJUIe-rkEyC7FZldV1sXx7zt_1ozR_T4-8L4epAuHPe7v4r9B-ZDxj7DQr0psI</recordid><startdate>20011001</startdate><enddate>20011001</enddate><creator>Waller, Anna</creator><creator>Pipkorn, David</creator><creator>Sutton, Karyn L.</creator><creator>Linderman, Jennifer J.</creator><creator>Omann, Geneva M.</creator><general>John Wiley & Sons, Inc</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>20011001</creationdate><title>Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands</title><author>Waller, Anna ; Pipkorn, David ; Sutton, Karyn L. ; Linderman, Jennifer J. ; Omann, Geneva M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5092-2e1d2675242fa3c8a1c3f59ce01c64bd57c30db9c4a25b21c49dd3f09abec5db3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>binding kinetics</topic><topic>Binding, Competitive</topic><topic>Calibration</topic><topic>flow cytometry</topic><topic>Flow Cytometry - methods</topic><topic>fluorescence calibration</topic><topic>Fluorescent Dyes - chemistry</topic><topic>Fluorescent Dyes - metabolism</topic><topic>G‐protein coupled receptor</topic><topic>Humans</topic><topic>Ligands</topic><topic>neutrophils</topic><topic>Neutrophils - metabolism</topic><topic>N‐formyl peptide receptor</topic><topic>Oligopeptides - chemistry</topic><topic>Oligopeptides - metabolism</topic><topic>Protein Binding</topic><topic>Reproducibility of Results</topic><topic>Spectrometry, Fluorescence - methods</topic><toplevel>online_resources</toplevel><creatorcontrib>Waller, Anna</creatorcontrib><creatorcontrib>Pipkorn, David</creatorcontrib><creatorcontrib>Sutton, Karyn L.</creatorcontrib><creatorcontrib>Linderman, Jennifer J.</creatorcontrib><creatorcontrib>Omann, Geneva M.</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 (New York, N.Y.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Waller, Anna</au><au>Pipkorn, David</au><au>Sutton, Karyn L.</au><au>Linderman, Jennifer J.</au><au>Omann, Geneva M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands</atitle><jtitle>Cytometry (New York, N.Y.)</jtitle><addtitle>Cytometry</addtitle><date>2001-10-01</date><risdate>2001</risdate><volume>45</volume><issue>2</issue><spage>102</spage><epage>114</epage><pages>102-114</pages><issn>0196-4763</issn><eissn>1097-0320</eissn><abstract>Background
Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor‐ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N‐formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands.
Methods
Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO‐NLFNYK‐tetramethylrhodamine.
Results
Calibration parameters were determined for six fluorescently‐labeled N‐formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO‐NLFNYK‐tetramethylrhodamine determined directly or in competition with CHO‐NLFNYK‐fluorescein were statistically identical.
Conclusions
Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated. Cytometry 45:102–114, 2001. © 2001 Wiley‐Liss, Inc.</abstract><cop>New York</cop><pub>John Wiley & Sons, Inc</pub><pmid>11590622</pmid><doi>10.1002/1097-0320(20011001)45:2<102::AID-CYTO1152>3.0.CO;2-Z</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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subjects | binding kinetics Binding, Competitive Calibration flow cytometry Flow Cytometry - methods fluorescence calibration Fluorescent Dyes - chemistry Fluorescent Dyes - metabolism G‐protein coupled receptor Humans Ligands neutrophils Neutrophils - metabolism N‐formyl peptide receptor Oligopeptides - chemistry Oligopeptides - metabolism Protein Binding Reproducibility of Results Spectrometry, Fluorescence - methods |
title | Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands |
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