The optimum conditions for the culture of cells from dissociated spleens were determined. Routinely, 10⁷ cells were seeded per ml of RPMI 1640 medium supplemented with 20% pre-tested foetal calf serum. For the assay of the immune response, cultures were supplemented with 30 µMolar mercaptoethanol.
The immune responses to sheep erythrocyte and bluetongue virus antigens were determined by the haemolytic plaque-forming cell assays described by Oellermann (1974) and Oellermann, Carter & Marx (1976a). The optimum sheep erythrocyte antigen concentration was 2 x 10⁶ erythrocytes per 10⁷ spleen cells and maximum IgM plaque-forming cells were detected after 4 days in culture. Successful stimulation of the immune response to bluetongue virus was achieved in spleen cell cultures from mice previously primed with bluetongue virus. The optimum antigen concentration was 30-40 ng bluetongue virus per 10⁷ spleen cells and the maximum plaque-forming cell response was observed after 4 days in culture.
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