A fluorometric assay for the quantitation of cell adherence to endothelial cells

Abstract

A rapid quantitative fluorometric assay was developed for analysis of leukocyte adherence to endothelial cells. In this method adherent monocyte and T cell lines are labeled with the fluorescent dye Calcein AM without affecting cell function. Following coincubation with endothelial cells and gentle washing to remove nonadhering cells, the relative fluorescence intensity of the adhering cells is determined with a fluorescence microtiter plate reader. Relative fluorescence intensity increases linearly with cell number over a wide range of concentrations. By comparison of fluorescence levels of adhering cells to a dilution series of labeled cells alone, the number of adhering cells can be determined. We compared this adherence assay with the 51Cr-labeling assay and found comparable adherence. However, we found the fluorescence assay to be more rapid as the use and special handling of radioactive material is eliminated. To monitor the reliability and reproducibility of this method, we followed the adherence of Calcein AM-labeled THP-1 cells, a human monocytic cell line, to human endothelial cells treated with interleukin-1.