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United States Department of Agriculture

Agricultural Research Service

Title: Five-Color Flow Cytometric Analysis of Swine Lymphocytes for Detection of Proliferation, Apoptosis, Viability, and Phenotype

Authors
item Waters, Wade
item Harkins, Kristi - IOWA STATE UNIV
item Wannemuehler, Michael - IOWA STATE UNIV.

Submitted to: Cytometry Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 14, 2002
Publication Date: N/A

Interpretive Summary: Diseases of pigs result in considerable loss of income for hog producers. An understanding of the basic function of immune cells isolated from pigs will aid development of disease intervention strategies. In this study, immune cells were collected from healthy pigs and stimulated with various stimulants. Expression of specific surface markers of responding cells was characterized using flow cytometric techniques. Advantages of this assay in comparison to other standard assays are described. Findings from this study provide a basis for the determination of the function of specific cells involved in the response of pigs to disease.

Technical Abstract: The objective of this study was to develop a method to simultaneously examine phenotype, proliferation, apoptosis, and death of antigen- stimulated porcine lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from pigs vaccinated with a Brachyspira hyodysenteriae bacterin. Once isolated, PBMC were stained with the vital fluorescent dye, PKH67, and cultured with or without B. hyodysenteriae whole cell sonicate antigen. Non-stimulated and B. hyodysenteriae-stimulated PBMC were harvested sequentially for flow cytometric analysis of proliferation (PKH67), apoptosis (Annexin V and 7AAD), viability (7AAD), and phenotype (e.g., CD3, CD4, CD8-alpha, CD8-beta, gamma/delta TCR, and/or IgM expression). 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 7AAD (viability). For analysis, gates were set on live (Annexin V**-, 7AAD**-), apoptotic (Annexin V**+, 7AAD**dim), and live/apoptotic (Annexin V**+/-, 7AAD**dim/-) cells and the phenotype of PBMC within these populations determined during the course of the in vitro response. Dead cells (e.g., 7AAD**bright) were excluded from the analysis. Application of this method for the determination of porcine lymphocyte subset proliferation is presented.

Last Modified: 8/21/2014
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