Isolation of bovine mammary lymphocytes for fluorescent activated flow cytometry

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.