Methods for simultaneous quantitative analysis of eosinophil and neutrophil adhesion and transmigration

Granulocytes play important roles in various inflammatory responses. The aim of this study was to develop in vitro methods to enable simultaneous analysis of eosinophil and neutrophil adhesion and transmigration in mixed granulocyte preparations. We used fibronectin-coated plates, with or without semipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)-5 and eotaxin or N-formyl-methionyl-leucyl-phenylalanine (fMLP), during incubation in the wells. Three different detergents: n-octyl-beta-D-glucopyranoside (OG), Triton-X-100 or N-cetyl-N,N, N-trimethylammonium bromide (CTAB), were tested for their ability to cause lysis of granulocytes with minimal effect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigenicity. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the most efficient lysing reagent. Cell-recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased in a time-dependent manner upon stimulation with IL-5 and eotaxin. Eosinophil adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigration upon activation with fMLP, reaching respective plateaux at 30 and 90 min. Our study shows that CTAB is an effective detergent for lysing granulocytes, yielding high and reproducible recovery rates of ECP and MPO. Measurement of ECP and MPO, as markers for cell counts, can therefore be used to quantify adhesive and transmigration properties of eosinophils and neutrophils in mixed granulocyte populations.