Submitted to: Journal of Immunological Methods Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 5, 2000
Publication Date: N/A
Interpretive Summary: Salmonella species of bacteria are a serious worldwide problem causing swine disease estimated to cost $100 million annually in the United States. Salmonella bacteria also are important human pathogens and are of great concern in food safety. Central to the study of porcine salmonellosis is the need to understand the potential effects of Salmonella infection on immune cell populations found in the blood and various tissues of the body. Thorough analysis of events surrounding infection requires testing of numerous animals at multiple time-points, a still somewhat time-consuming and labor-intensive project. This study describes a rapid and reproducible method for identification of pig immune cell populations using flow cytometry. This new method uses whole blood, instead of partially purified cells, and allows the simultaneous addition of all but one of the required components. In the final step, cells are added to a test tube containing 1.5% formalin for lysis and fixation. This new procedure requires no wash steps. Total sample preparation time is less than 2 hours. Eventual beneficiaries of this test are the American consumer since new diagnostic and intervention strategies for salmonellosis will allow a continued supply of inexpensive, wholesome pork and pork products.
Technical Abstract: The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 ul of an appropriate dilution of four different antibodies (40 ul total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 deg C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 minutes. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 deg C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 hours. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.