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United States Department of Agriculture

Agricultural Research Service

Title: Characterization of Mitogen-Stimulated Porcine Lymphocytes Using Multicolorflow Cytometry

Authors
item Dorn, A - IOWA STATE UNIVERSITY
item Waters, Wade
item Byers, V - IOWA STATE UNIVERSITY
item Pesch, Bruce
item Wannemuehler, M - IOWA STATE UNIVERSITY

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 28, 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: Stimulation of lymphocyte proliferation using mitogens or specific antigens is a frequently used assay to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [**3H]-thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multi-color flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured by both [**3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [**3H]-thymidine incorporation data alone, it was observed that PBMCs stimulated with either Concanavalin A (Con A), Phytohemagglutinin (PHA) or Pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was determined. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4**+CD8(**+, CD4**-CD8(**hi, CD4CD8(**lo, and gd TCR**+ cells. PWM stimulation induced responses by CD4**+CD8(**+, CD4**-CD8(**hi but not by CD4**-CD8(**lo or gd TCR**+ cells. Con A stimulation resulted in a sustained proliferation of CD8(**hi cells over the 5 day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM**+ population (e.g., B cells).

Last Modified: 12/18/2014
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