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

Granulocytes play important roles in various inflammatory responses. The ai m of this study was to develop in vitro methods to enable simultaneous anal ysis of eosinophil and neutrophil adhesion and transmigration in mixed gran ulocyte preparations. We used fibronectin-coated plates, with or without se mipermeable inserts, to measure adhesion and transmigration. Granulocytes, from healthy blood donors, were stimulated with either interleukin (IL)-5 a nd eotaxin or N-formyl-methionyl-leucyl-phenylalanine (fMLP), during incuba tion in the wells. Three different detergents: n-octyl-beta-D-glucopyranosi de (OG), Triton-X-100 or N-cetyl-N,N,N-trimethylammonium bromide (CTAB), we re tested for their ability to cause lysis of granulocytes with minimal eff ect on eosinophil cationic protein (ECP) and myeloperoxidase (MPO) antigeni city. These two proteins were used for quantitative analysis of eosinophil and neutrophil adhesion/transmigration, and CTAB was the must efficient lys ing reagent. Cell-recovery rates, based on ECP/MPO measurements, were > 95% in both assays. The adhesion and transmigration of eosinophils increased i n a time-dependent manner upon stimulation with IL-5 and eotaxin. Eosinophi l adhesion reached a plateau at 90 min of incubation and transmigration at 240 min. Neutrophils displayed a similar pattern of adhesion and transmigra tion upon activation with fMLP, reaching respective plateaux at 30 and 90 m in. Our study shows that CTAB is an effective detergent for lysing granuloc ytes, yielding high and reproducible recovery rates of ECP and MPO. Measure ment of ECP and MPO, as markers for cell counts, can therefore be used to q uantify adhesive and transmigration properties of eosinophils and neutrophi ls in mixed granulocyte populations.