Journal of Immunological Methods, 100 (1987) 223-233 223 Elsevier JIM 04376 Functional neutrophils from long-term murine bone marrow cell cultures P.H. Hart 1, L.K. Spencer l, P.A. Kenny 1, A.F. Lopez 2, p.j. McDonald 1 and J.J. Finlay-Jones 1 t Department of Clinical Microbiology, School of Medicine, Flinders University of S.A., Bedford Park, S.A. 5042, and 2 Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, S.A. 5000, Australia (Received 2 February 1987, accepted 23 February 1987) Murine bone marrow cell cultures that had been established for up to 26 weeks were harvested each week and found to provide functional neutrophils. Leukocytes harvested from the cultures were enriched for neutrophils using discontinuous PercoU density gradients. These cells mounted a chemiluminescence response to Proteus mirabilis in the presence of normal mouse serum (NMS). They killed several NMS-opsonised bacterial species, an activity that was blocked by a monoclonal antibody to the C3 receptor of mouse neutrophils. Cultured bone marrow neutrophils expressed both Fc and C3 receptors. C3 receptor expression could be augmented by exposure to the chemotactic peptide f-Met-Leu-Phe. We conclude that murine bone marrow cell cultures provide a useful source of functional neutrophils, and that their productivity can be sustained in long-term culture. As their receptor expression can be augmented from the resting state by exogenous stimuli, they represent a useful cell source in studies of neutrophil activation. Key words: Bone marrow cell culture: Neutrophii; C3 receptor: f-Met-Leu-Phe; (Mouse) 1. Inb'oduction Human peripheral venous blood provides a source of readily purified neutrophils. With 1-2 h work and by use of density gradients (BiSyum, 1976), approximately 2 x 106 neutrophils can be obtained per ml of blood. This is not so with mice. Greater than 1.5 ml of blood is rarely obtainable from a normal mouse. The yield of leukocytes/ml of venous blood following hypotonlc lysis of erythrocytes is approximately 2 x 106. However, neutrophils comprise only 6%-38% of the leuko- Correspondence to: J.J. Finlay-Jones, Department of Clini- cal Microbiology, School of Medicine, Flinders University of S.A., Bedford Park, S.A. 5042, Australia. cytes in the peripheral blood of normal mice (Rus- sell et al., 1951). Inflammatory agents can stimulate neutrophil influx into the peritoneal cavity, providing an alternative source of murine cells (Baron and Proctor, 1982; Brummer and Stevens, 1983; Zim- merli et al., 1984). From each of the peritoneal cavities of male BALB/c mice, given an in- flammatory stimulus 3.5 h previously, approxi- mately 107cells can be harvested, comprising about two thirds neutrophils (Hart et al., 1985). The increased yield of neutrophils per mouse from the peritoneal cavity provides a more useful source of cells for study. However, neutrophils obtained from a site of acute inflammation may be func- tionally different from peripheral blood neu- trophils (Zimmerli et al., 1984). 0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)