Five-color flow cytometric analysis of swine lymphocytes for detection of proliferation, apoptosis, viability, and phenotype.
BACKGROUND
The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen-stimulated porcine lymphocytes.


METHODS
Peripheral blood mononuclear cells (PBMCs) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin.


RESULTS
Once isolated, PBMCs were stained with the fluorescent membrane intercalating dye, PKH67, and cultured with or without B. hyodysenteriae whole-cell sonicate antigen. Serial samples of nonstimulated and B. hyodysenteriae-stimulated PBMCs were harvested for flow cytometric analysis. Fluorochrome excitation was performed with spatially separated air-cooled argon and red helium neon laser beams. Five-color analysis included signal detection of PKH67 (proliferation), phycoerythrin (cell surface antigen), Texas Red phycoerythrin tandem (cell surface antigen), allophycocyanin (annexin V), and 7-amino-actinomysin D (7AAD; viability). For analysis, gates were set on live (annexin V(-), 7AAD(-)), intact apoptotic (annexin V(+), 7AAD(dim)), and live plus intact apoptotic (annexin V(+/-), 7AAD(dim/-)) cells, and the phenotypes of PBMCs within these populations were determined during the course of the in vitro response. Dead cells (i.e., 7AAD(bright)) were excluded from the analysis.


CONCLUSIONS
Application of this method for the determination of porcine lymphocyte subset proliferation is presented.
Waters
W Ray
WR
United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa 50010-0070, USA. rwaters@nadc.ars.usda.gov
Harkins
Kristi R
KR
Wannemuehler
Michael J
MJ
D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D000942:Antigens, Bacterial / Q000276:immunology; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D002455:Cell Division; D002470:Cell Survival; D003116:Color; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000032:analysis; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D016130:Immunophenotyping / Q000379:methods*; D008213:Lymphocyte Activation; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology / Q000473:pathology*; D009930:Organic Chemicals; D010641:Phenotype; D013552:Swine; D014611:Vaccination
D017304:Annexin A5; D000942:Antigens, Bacterial; D015415:Biomarkers; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C451241:PKH67; D003609:Dactinomycin; C025942:7-aminoactinomycin D