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 microl of an appropriate dilution of four different antibodies (40 microl 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 degrees 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 min. 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 degrees 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 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.
Stabel
T J
TJ
United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Road, Ames, IA 50010, USA. tstabel@nadc.ars.usda.gov
Bolin
S R
SR
Pesch
B A
BA
Rahner
T E
TE
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis*; D001773:Blood Cells / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D007962:Leukocytes / Q000276:immunology; D015203:Reproducibility of Results; D013552:Swine / Q000097:blood* / Q000276:immunology*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface