Submitted to: American Association of Immunologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: May 16, 2000
Publication Date: N/A
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 ml of an appropriate dilution of four different antibodies (40 ml 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 diluted in 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. Samples may be acquired 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. 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 minimum amount of time.