A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates.
We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.
Weetall
M
M
Novartis Pharmaceuticals, Summit, New Jersey 07901, USA. marla.weetall@pharma.novartis.com
Hugo
R
R
Friedman
C
C
Maida
S
S
West
S
S
Wattanasin
S
S
Bouhel
R
R
Weitz-Schmidt
G
G
Lake
P
P
D018926:Anti-Allergic Agents / Q000037:antagonists & inhibitors / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002448:Cell Adhesion; D019040:E-Selectin / Q000378:metabolism; D005189:False Positive Reactions; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D039041:Integrin alpha4beta1; D016023:Integrins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008024:Ligands; D008970:Molecular Weight; D009010:Monte Carlo Method; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D016201:Receptors, Lymphocyte Homing / Q000037:antagonists & inhibitors / Q000378:metabolism; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D018926:Anti-Allergic Agents; D019040:E-Selectin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D039041:Integrin alpha4beta1; D016023:Integrins; D008024:Ligands; D009844:Oligosaccharides; D016201:Receptors, Lymphocyte Homing; D019010:Vascular Cell Adhesion Molecule-1; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester