Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies

Robert E. Truax From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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Mary D. Powell From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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Marilyn A. Dietrich From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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Dennis D. French From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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John A. Ellis From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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Mark J. Newman From the Department of Veterinary Microbiology and Parastiology (Truax, Powell, Dietrich, Newman) School of Veterinary Medicine and the Department of Veterinary Science (French), Louisiana State University, Baton Rouge, LA 70803, and the Department of Veterinary Sciences (Ellis), Wyoming State Veterinary Laboratory, Laramie, WY 82070.

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Summary

A simple cryogenic technique for preserving bovine buffy coat leukocytes was developed. This was coupled with a variation of the standard discontinuous gradient technique to purify mononuclear cells that retained immunologic function. The total number of mononuclear cells recovered from cyropreserved samples were only 87 to 42% of those recovered from freshly obtained blood samples. However, the functional capabilities of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measured, 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 leukocytes that responded to irradiated B abortus. These antigen-specific responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surface IgM on B lymphocytes was also evaluated. Flow cytometric analysis of 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 processing of large numbers of samples with the ability to assay various immune functions at a later time.

Summary

A simple cryogenic technique for preserving bovine buffy coat leukocytes was developed. This was coupled with a variation of the standard discontinuous gradient technique to purify mononuclear cells that retained immunologic function. The total number of mononuclear cells recovered from cyropreserved samples were only 87 to 42% of those recovered from freshly obtained blood samples. However, the functional capabilities of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measured, 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 leukocytes that responded to irradiated B abortus. These antigen-specific responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surface IgM on B lymphocytes was also evaluated. Flow cytometric analysis of 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 processing of large numbers of samples with the ability to assay various immune functions at a later time.

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