Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands
dc.contributor.author | Waller, Anna | en_US |
dc.contributor.author | Pipkorn, David | en_US |
dc.contributor.author | Sutton, Karyn L. | en_US |
dc.contributor.author | Linderman, Jennifer J. | en_US |
dc.contributor.author | Omann, Geneva M. | en_US |
dc.date.accessioned | 2006-04-19T13:46:18Z | |
dc.date.available | 2006-04-19T13:46:18Z | |
dc.date.issued | 2001-10-01 | en_US |
dc.identifier.citation | Waller, Anna; Pipkorn, David; Sutton, Karyn L.; Linderman, Jennifer J.; Omann, Geneva M. (2001)."Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands." Cytometry 45(2): 102-114. <http://hdl.handle.net/2027.42/34699> | en_US |
dc.identifier.issn | 0196-4763 | en_US |
dc.identifier.issn | 1097-0320 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/34699 | |
dc.identifier.uri | http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=11590622&dopt=citation | en_US |
dc.description.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. | en_US |
dc.format.extent | 446804 bytes | |
dc.format.extent | 3118 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | |
dc.publisher | John Wiley & Sons, Inc. | en_US |
dc.subject.other | Life and Medical Sciences | en_US |
dc.subject.other | Cell & Developmental Biology | en_US |
dc.title | Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands | en_US |
dc.type | Article | en_US |
dc.rights.robots | IndexNoFollow | en_US |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | en_US |
dc.subject.hlbtoplevel | Health Sciences | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.peerreviewed | Peer Reviewed | en_US |
dc.contributor.affiliationum | Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan | en_US |
dc.contributor.affiliationum | Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan | en_US |
dc.contributor.affiliationum | Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan | en_US |
dc.contributor.affiliationum | Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan | en_US |
dc.contributor.affiliationum | Departments of Biological Chemistry and Surgery, University of Michigan Medical School and Veteran's Administration Medical Center, Ann Arbor, Michigan ; Research Service (11R), Veteran's Administration Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105 | en_US |
dc.identifier.pmid | 11590622 | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/34699/1/1152_ftp.pdf | en_US |
dc.identifier.doi | http://dx.doi.org/10.1002/1097-0320(20011001)45:2<102::AID-CYTO1152>3.0.CO;2-Z | en_US |
dc.identifier.source | Cytometry | en_US |
dc.owningcollname | Interdisciplinary and Peer-Reviewed |
Files in this item
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.