Conditioned medium from macrophage cell lines supports the single-cell growth of hybridomas

The aim of this study was to establish whether conditioned medium (CM) from macrophage cell lines would support the growth of hybridomas under conditions commonly used in hybridization experiments and in cloning of antigen-specific hybridomas. The ability of CM from macrophage cell lines J774, WEHI 274, WEHI 265, and PU 5 to support single-cell growth during cloning was compared with CM from cultures of resident mouse peritoneal cells, EL 4 mouse thymoma cells, L929 mouse fibrosarcoma, and feeder layers of resident peritoneal cells. CM from J774, L929, and resident peritoneal cells supported single-cell growth at the same level as the macrophage feeder layer. J774 and L929 CM were most effective at a final concentration of 25% with fresh medium supplemented with 20% fetal calf serum (FCS). The ability of J774 CM to support hybridoma growth was increased by prior stimulation with LPS but not PMA. CM from LPS-stimulated J774 cells used in fusion experiments resulted in increased numbers of hybridomas compared with those obtained with macrophage feeder layers.