| An arrayed high-content chemotaxis assay for patient diagnosis. | |
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MedLine Citation:
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PMID: 20953490 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases. |
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Authors:
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Erwin Berthier; Jill Surfus; James Verbsky; Anna Huttenlocher; David Beebe |
Publication Detail:
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Type: In Vitro; Journal Article; Research Support, Non-U.S. Gov't Date: 2010-10-18 |
Journal Detail:
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Title: Integrative biology : quantitative biosciences from nano to macro Volume: 2 ISSN: 1757-9708 ISO Abbreviation: Integr Biol (Camb) Publication Date: 2010 Nov |
Date Detail:
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Created Date: 2010-11-02 Completed Date: 2011-02-18 Revised Date: 2011-08-22 |
Medline Journal Info:
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Nlm Unique ID: 101478378 Medline TA: Integr Biol (Camb) Country: England |
Other Details:
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Languages: eng Pagination: 630-8 Citation Subset: IM |
Affiliation:
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Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin, 1111 Highland Av, Madison 53705, Wisconsin, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Cell Polarity Cell Tracking / instrumentation, methods Chemotaxis, Leukocyte* / drug effects Equipment Design HL-60 Cells Humans Immunologic Deficiency Syndromes / diagnosis, genetics, immunology Immunologic Tests / instrumentation, methods* Infant Microfluidic Analytical Techniques / instrumentation, methods* Models, Biological Mutation N-Formylmethionine Leucyl-Phenylalanine / pharmacology Neutrophils / drug effects, immunology, physiology rac GTP-Binding Proteins / genetics |
| Chemical | |
Reg. No./Substance:
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59880-97-6/N-Formylmethionine Leucyl-Phenylalanine; EC 3.6.1.-/rac2 GTP-binding protein; EC 3.6.5.2/rac GTP-Binding Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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