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Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies
R E Truax1, M D Powell, M A Dietrich
1Department of Veterinary Microbiology and Parastiology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803.
Insights
A new cryogenic method effectively preserves bovine buffy coat leukocytes, maintaining their immunologic function and antigen-specific responses after cryopreservation. This technique allows for later analysis of immune cell capabilities.
Area of Science:
- Veterinary Immunology
- Cryobiology
- Cellular Immunology
Background:
- Cryopreservation of leukocytes is crucial for preserving immune function.
- Standard methods may compromise cell viability and function.
- A simple, effective method for bovine leukocytes is needed.
Purpose of the Study:
- To develop a simple cryogenic technique for preserving bovine buffy coat leukocytes.
- To assess the immunologic function of cryopreserved mononuclear cells.
- To evaluate the retention of antigen-specific responses and cell surface markers.
Main Methods:
- Developed a simple cryogenic technique for bovine buffy coat.
- Used a modified discontinuous gradient to purify mononuclear cells.
- Assessed cell function via polyclonal proliferation assays and antigen-specific responses to Brucella abortus.
- Analyzed cell surface marker expression (CD2, CD4, CD8, IgM) using flow cytometry.
Main Results:
- Cryopreservation yielded 42-87% of mononuclear cells compared to fresh samples.
- Mononuclear cells retained normal polyclonal proliferative responses to mitogens.
- Antigen-specific responses to Brucella abortus were preserved.
- Flow cytometry showed no alteration in the relative proportions of T-lymphocyte and B-lymphocyte subpopulations.
Conclusions:
- The developed cryogenic technique is simple and effective for preserving bovine buffy coat leukocytes.
- Cryopreserved cells retain crucial immunologic functions, including proliferation and antigen-specific responses.
- This method facilitates future immune function assays on large sample sets.
Abstract:
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.