A FLUOROMETRIC ASSAY FOR THE QUANTITATION OF CELL ADHERENCE TO ENDOTHELIAL-CELLS

A rapid quantitative fluorometric assay was developed for analysis of leukocyte adherence to endothelial cells. In this method adherent mono cyte and T cell lines are labeled with the fluorescent dye Calcein AM without affecting cell function. Following coincubation with endotheli al cells and gentle washing to remove nonadhering cells, the relative fluorescence intensity of the adhering cells is determined with a fluo rescence microtiter plate reader. Relative fluorescence intensity incr eases linearly with cell number over a wide range of concentrations. B y comparison of fluorescence levels of adhering cells to a dilution se ries of labeled cells alone, the number of adhering cells can be deter mined. We compared this adherence assay with the Cr-51-labeling assay and found comparable adherence. However, we found the fluorescence ass ay to be more rapid as the use and special handling of radioactive mat erial is eliminated. To monitor the reliability and reproducibility of this method, we followed the adherence of Calcein AM-labeled THP-1 ce lls, a human monocytic cell line, to human endothelial cells treated w ith interleukin-1.