A NEW ADHESION ASSAY USING BUOYANCY TO REMOVE NONADHERENT CELLS

A new adhesion assay was developed that utilizes buoyancy, rather than washing or centrifugation, to remove non-adherent cells. Biotinylated cells were added to wells containing cell monolayers or purified protein substrates. Non-adherent cells were then removed by floatation on a dense Percoll solution. The adherent cells were fixed tightly to the plate with a Percoll/glutaraldehyde fixative and quantitated by streptavidin: horseradish peroxidase chemistry. In a side-by-side comparison of buoyancy and washing assays, the buoyancy method detected B16F10 binding to purified fibronectin at a 4-fold lower fibronectin concentration and human umbilical vein endothelia cell (HUVEC) binding to laminin at a 10-fold lower laminin concentration than did washing assays. In cell to cell adhesion assays, the buoyancy method was able to detect significantly greater binding of mononuclear leukocytes and KM12-L4 colon carcinoma cells to IL-1 beta treated human umbilical vein endothelial cells (HUVEC). The binding of human promyelocytic leukemia HL60 cells to control and IL-1 beta treated HUVEC was the same (approximately 60%) with the buoyancy method, while a washing assay demonstrated 8-fold higher binding (51% vs. 6%) of HL60 on IL-1 beta treated cells. The buoyancy assay is useful for detecting weak cell to protein adhesion and may be useful for detecting cell to cell adhesion when background binding is sufficiently low.