CRYOPRESERVATION OF BOVINE BUFFY COAT LEUKOCYTES FOR USE IN IMMUNOLOGICAL STUDIES

A simple cryogenic technique for preserving bovine buffy coat leukocyt es was developed. This was coupled with a variation of the standard di scontinuous gradient technique to purify mononuclear cells that retain ed immunologic function. The total number of mononuclear cells recover ed from cyropreserved samples were only 87 to 42% of those recovered f rom freshly obtained blood samples. However, the functional capabiliti es of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measu red, using a titration of mitogen concentrations, and were found to be normal, compared with those of cells isolated from fresh blood. Blood samples collected after vaccination with Brucella abortus contained l eukocytes that responded to irradiated B abortus. These antigen-specif ic responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surfa ce IgM on B lymphocytes was also evaluated. Flow cytometric analysis o f fresh and cryopreserved mononuclear cell preparations indicated that the relative proportions of different subpopulations were not altered . The technical simplicity of our cryopreservation system will allow p rocessing of large numbers of samples with the ability to assay variou s immune functions at a later time.