Development of a high capacity microassay for measurement of neutrophil adhesion

A high capacity semiautomated assay for neutrophil adhesion was developed utilizing the 96 well microtiter plate format. Optimal adhesion occurred with about 150 microliters/well of neutrophils at 5 X 10(6) cells/ml in tissue culture plates that had been precoated with 5% serum. Optimal incubation times were 10 min for f-Met-Leu-Phe-treated cells and 20 min for A23187 or phorbol myristate acetate stimulation. Optimal washing occurred after three washes with a Cetus Pro/pette pump. Adhesion could be effectively blocked by the inhibitors of cellular protein kinase C, an enzyme known to be necessary for the occurrence of neutrophil adhesion.