Isolation and purification of functional bovine lung mast cells (BLMCs)

Purified pulmonary mast cells were obtained from bovine lung using a combination of enzymatic digestion of tissue, density gradient centrifugation using Percoll, and centrifugal elutriation. In the initial procedure, lung tissue was enzymatically digested with collagenase, hyaluronidase, protease and elastase in three 30 min incubations at 37 degrees C. Monodispersed cell suspensions contained between 2 and 6% mast cells. Further purification of these mast cells by Percoll gradients and elutriation consistently yielded mast cells of > 90% purity. These cells were morphologically intact, viable and functional, as determined by histamine release evoked by secretagogue challenge. Incubation of BLMCs with Pasteurella haemolytica A1 culture supernate containing leucotoxin (LCT) alone, resulted in increased histamine release compared to controls. LCT also potentiated calcium ionophore (CaI)-induced histamine release from BLMCs.