Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination

A method for quantitating the number of polymorphonuclear leukocytes (PMNs) adhering to endothelial cells in vitro is presented. Confluent human umbilical vein endothelial cells cultured in 24-well multiplates and treated with tumor necrosis factor alpha (TNF alpha) were incubated subsequently with PMNs which adhere to the endothelial cell as a function of TNF alpha concentration. Adherent PMNs and endothelial cells were proteolytically dissociated from the multiwell and, using an electronic particle counter, the number of endothelial cells and PMNs were determined simultaneously on the basis of size. The average number of PMNs adhering per endothelial cell was then calculated. The method is rapid and precise and offers an alternative to both tedious microscopic counting and the hazardous radiolabeling of PMNs.