The flow cytometric evaluation of phagocytosis by equine peripheral blood neutrophils and pulmonary alveolar macrophages.

Flow cytometry was used to assess the phagocytosis of fluorescent-labelled bacteria by equine peripheral blood neutrophils and pulmonary alveolar macrophages. Cell populations were prepared from venous blood following ammonium chloride lysis and from washed bronchoalveolar lavage derived samples. Discrete clusters of cells, corresponding to different leucocyte groups, were readily identified on the basis of differing light scattering properties and could thus be discriminated, negating the need for prior cell separation. Cells able to associate with fluorescent-labelled bacteria (by attachment to the cell membrane or by internalization within the cell) acquired increased fluorescence and were readily differentiated from cells unable to interact with bacteria. The fluorescence of bacteria attached to the cell surface was quenched by the addition of trypan blue or counterstained by the addition of ethidium bromide to the assay, thus permitting identification of cells which were able to internalize bacteria.