A fluorescent cellular adhesion assay using insect cell produced human VCAM1.
Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counterreceptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.
Stoltenborg
J K
JK
Department of Cardiovascular Diseases, Du Pont Merck Pharmaceutical Company, Wilmington, DE 19880-0400.
Tsao
P W
PW
George
H J
HJ
Bouchard
P J
PJ
Wexler
E J
EJ
Hausner
E A
EA
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D005434:Flow Cytometry; D006801:Humans; D016029:Receptors, Very Late Antigen / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D018411:Spodoptera; D014162:Transfection; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1
D015815:Cell Adhesion Molecules; D016029:Receptors, Very Late Antigen; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1