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
Hauptverfasser: Waller, Anna, Pipkorn, David, Sutton, Karyn L., Linderman, Jennifer J., Omann, Geneva M.
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container_end_page 114
container_issue 2
container_start_page 102
container_title Cytometry (New York, N.Y.)
container_volume 45
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
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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. 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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. 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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 &amp; Sons, Inc</pub><pmid>11590622</pmid><doi>10.1002/1097-0320(20011001)45:2&lt;102::AID-CYTO1152&gt;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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