Abstract
A detailed methodology is described for the isolation of T lymphocytes from bovine mammary gland secretions for flow cytometry. Mammary gland secretions are collected and centrifuged to separate the leukocytes from the mammary supernatant. The leukocytes are purified using a density gradient and diluted for fluorescent staining. Using tri-color fluorescent staining, the T lymphocytes are identified by monoclonal antibodies and stained with secondary antibodies. Flow cytometry is used to quantify the cellular subpopulations of the T lymphocytes. Proportional and statistical analysis of the flow cytometric data is conducted with computer software, CELLQuest and SAS.
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Marr JC, Lyon JD, Roberson JR, Lupher M, Davis WC, Bohach GA (1993). Characterization of novel type C staphylococcal enterotoxins: biological and evolutionary implications. Infect Immun 61: 4254-4262.
Ferens WA, Davis WC, Hamilton MJ, Park YH, Deobald CF, Fox LK, Bohach GA (1998). Activation of bovine lymphocytes by staphylococcal enterotoxin C. Infect Immun 66: 573-580.
Park YH, Fox LK, Hamilton MJ, Davis WC (1992). Bovine mononuclear leukocyte subpopulations in peripheral blood and mammary gland secretions during lactation. J Dairy Sci 75: 998-1006.
Davis WC, Davis JE, Hamilton MJ (1995). Use of monoclonal antibodies and flow cytometry to cluster and analyze leukocyte differentiation molecules. In: Davis WC (ed), Methods in molecular biology, Vol. 45: Monoclonal antibody protocols. Totowa, NJ: Humana Press, Inc., pp 149-167.
Baldwin CL, Teale AJ, Naessens J, Goddeeris BM, MacHugh ND, Morrison WI (1986). Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: Similarity to lymphocytes defined by human T4 and murine L3T4. J Immunol 136: 4385-4391.
Bembridge GP, Parsons KR, Sopp P, MacHugh ND, Howard CJ (1993). Comparison of monoclonal antibodies with potential specificity for restricted isoforms of the leukocyte common antigen (CD45R). Vet Immunol Immunopathol 39: 129-136.
Naessens J, Sileghem M, MacHugh N, Davis WC, Toye P (1992). Selection of BoCD25 monoclonal antibodies by screening mouse L cells transfected with the bovine p55-interleukin-2 (IL-2) receptor gene. Immunology 76: 305-309.
Monoclonal antibody 7C2B obtained from Dr. WC Davis at Washington State University.
Morrison WI, Davis WC (1991). Differentiation antigens expressed predominantly on CD4-CD8-T lymphocytes (WC1, WC2). Vet Immunol Immunopathol 27: 71-76.
Park YH, Fox LK, Hamilton MJ, Davis WC (1993). Supression of proliferative response of BoCD4+ T lymphocytes by activated BoCD8+ T lymphocytes in the mammary gland of cows with Staphylococcus aureus mastitis. Vet Immunol Immunopathol 36: 137-151.
Davis WC, Splitter GS (1991). Bovine CD2 (BoCD2). Vet Immunol Immunopathol 27: 43-50.
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Ebling, T.L., Fox, L.K. & Byrne, K.M. Isolation of bovine mammary lymphocytes for fluorescent activated flow cytometry. Methods Cell Sci 22, 239–245 (2000). https://doi.org/10.1023/A:1009812629496
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DOI: https://doi.org/10.1023/A:1009812629496