Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands

Autor: Geneva M. Omann, Jennifer J. Linderman, Karyn L. Sutton, David Pipkorn, Anna Waller
Rok vydání: 2001
Předmět:
Zdroj: Cytometry. 45:102-114
ISSN: 1097-0320
0196-4763
Popis: 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 CHONLFNYK-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. Key terms: neutrophils; fluorescence calibration; binding kinetics; G-protein coupled receptor; N-formyl peptide receptor; flow cytometry
Databáze: OpenAIRE
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