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Cytometry A. 2004 Jan;57(1):39-44.

Hollow-fiber assay for ligand-mediated cell adhesion.

Author information

1
Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia. r.nordon@unsw.edu.au

Abstract

BACKGROUND:

The investigation of receptor-ligand interactions in the cellular context presents significant technical challenges, first, to immobilize the ligand in a manner that preserves functional properties and, second, to relate ligand properties to cell adhesion and other cellular processes.

METHODS:

Ligand-mediated cell adhesion was characterized by the development of a cellulose hollow-fiber adhesion assay in which ligand (protein A) was immobilized onto the cellulose membrane as a recombinant fusion protein containing a cellulose-binding domain affinity tag. Modules containing single cellulose hollow fibers were connected to a micro-flow system for cell deposition and detachment with fluid shear stress. The cell adhesion process that occurred inside a segment of hollow fiber was observed in real time by using an inverted microscope equipped with a CCD camera and digital frame grabber. Image analysis software was developed to count cells and record digital images.

RESULTS:

Cell adhesion strength was characterized by counting the number of cells that were detached by application of fluid shear stress with values that ranged from 2.3 to 185 dyne/cm2. The median shear stress of detachment of KG1a cells was directly related to the duration of membrane contact and the amount of immobilized monoclonal antibody (anti-CD34).

CONCLUSIONS:

The hollow-fiber assay provides a general method to determine functional properties of molecular domains that interact with cell surface receptors and markers.

PMID:
14699604
DOI:
10.1002/cyto.a.10091
[Indexed for MEDLINE]
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