Collection and cultivation in vitro of equine mammary macrophages

Equine macrophages were obtained from female Shetland ponies by injection of Escherichia coli lipopolysaccharide through the lactiferous ducts of the mammary gland. After 6 to 11 days, balanced salt solution was injected into the mammary gland to wash out accumulated cells. Harvested cells contained a mixture of macrophages, lymphocytes, and neutrophils, with the majority of the cells of mononuclear type. In culture, cells adherent after 24 hours were characterized as macrophages by morphologic features, nonspecific esterase staining, and by the presence of complement and immunoglobulin receptors. These cultures were grown to a variety of culture media. A basal medium, consisting of 15% equine serum and 10% bovine fetal serum in conjunction with RPMI 1640 medium containing 20 mM HEPES buffer, was the most effective for maintaining spreading and adhesion of cells. Conditioned medium from mouse fibroblast cultures (L cells), added at 30% to the basal medium, improved cell monolayers by reducing giant cell formation and prolonging cell adhesion.