CORRECTION FOR LABEL LEAKAGE IN FLUOROMETRIC ASSAYS OF CELL-ADHESION

Current flourescence-based adhesion assays that use a 96-well plate fo rmat rely on the assumption that the fluorescent label does not signif icantly leak from the cells. Thus we evaluated a calcein-based, in vit ro adhesion assay in 96-well plates using five different types of leuk ocytes (HL60 cells, human neutrophils, rat neutrophils, mouse progenit or T cells and EL4 cells). Each cell type leaked calcein at a differen t rate, with the highest rates found for rat neutrophils and progenito r T cells. which lost as much as 20%-40% of the label within 90 min, t he time required to complete the assay. Thus, we developed a procedure to measure the dye leakage rate during the assay in order to obtain a correction factor, which was then used to calculate the ''true'' numb er of adherent cells. Data for the adhesion of FTF1 cells to endotheli al monolayers, after correction for calcein leakage, deviated less tha n 10% of adhesion data obtained with a well-established Cr-51-based as say.