Hollow-fiber assay for ligand-mediated cell adhesion.
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.
Nordon
Robert E
RE
Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia. r.nordon@unsw.edu.au
Shu
Alan
A
Camacho
Fernando
F
Milthorpe
Bruce K
BK
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D001681:Biological Assay; D002448:Cell Adhesion / Q000502:physiology*; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D002482:Cellulose / Q000737:chemistry; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007951:Leukemia, Myeloid; D008024:Ligands; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013314:Stress, Mechanical
D000911:Antibodies, Monoclonal; D008024:Ligands; D011993:Recombinant Fusion Proteins; D002482:Cellulose