A simple and rapid flow cytometric method for detection of porcine cell surface markers

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 mul of an appropriate dilution of four different antibodies (40 mul total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenie nce, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degreesC. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temp erature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 mi 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 degreesC 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 popula tions, and total number of granulocytes identified using this method were c omparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells w as lower than standard values. Total assay time from receipt of blood to ac quisition of data by flow cytometry required less than 2 h. This modified a ssay was shown to be simple, reliable, and useful for screening large numbe rs of porcine samples in a minimal period of time. (C) 2000 Elsevier Scienc e BN. All rights reserved.