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Updated: Aug 8, 2026
Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
Published on: April 23, 2010
Characterization of mitogen-stimulated porcine lymphocytes using a stable fluorescent dye (PKH2) and multicolor flow
A D Dorn1, W R Waters, V M Byers
1Veterinary Medical Research Institute, Iowa State University, 1802 Elwood Drive, Ames, IA 50011, USA. adorn@iastate.edu
Insights
This study introduces PKH2 dye and flow cytometry to measure porcine lymphocyte proliferation, identifying specific responding cell subsets to mitogens like Con A and PHA.
Area of Science:
- Immunology
- Cell Biology
- Veterinary Science
Background:
- Assessing immune responsiveness often involves lymphocyte proliferation assays, such as [3H]-thymidine incorporation.
- However, traditional methods offer limited insight into the proliferation of specific lymphocyte subsets.
- Porcine immune studies require precise methods to analyze lymphocyte subpopulations.
Purpose of the Study:
- To introduce and validate a novel flow cytometry-based assay using PKH2 dye for measuring porcine lymphocyte proliferation.
- To differentiate the proliferative responses of distinct porcine lymphocyte subsets to various mitogens.
- To provide a detailed analysis of lymphocyte subset contributions to mitogen-induced proliferation in pigs.
Main Methods:
- Porcine peripheral blood mononuclear cells (PBMCs) were stimulated with mitogens (Concanavalin A, Phytohemagglutinin, Pokeweed Mitogen).
- Lymphocyte proliferation was assessed using both traditional [3H]-thymidine incorporation and a novel flow cytometric assay with PKH2 fluorescent dye.
- Multicolor flow cytometry was employed to analyze the proliferation of specific lymphocyte subsets (e.g., CD4, CD8, gammadelta T cells).
Main Results:
- The PKH2 dye and flow cytometry assay successfully measured mitogen-induced proliferation of porcine lymphocyte subpopulations.
- Concanavalin A and Phytohemagglutinin primarily stimulated CD4(+)CD8(+), CD4(-)CD8alpha(hi), CD4(-)CD8alpha(lo), and gammadelta TCR(+) cells.
- Pokeweed Mitogen stimulated CD4(+)CD8(+) and CD4(-)CD8alpha(hi) cells, but not CD4(-)CD8alpha(lo) or gammadelta TCR(+) cells.
- Proliferative responses varied between mitogens, with Con A showing sustained CD8alpha(hi) proliferation and PHA peaking at day 3.
- B cells (IgM+) exhibited minimal proliferative responses to the tested mitogens.
Conclusions:
- PKH2 dye combined with multicolor flow cytometry is an effective tool for analyzing porcine lymphocyte subset proliferation.
- This method provides a more granular understanding of immune responses compared to traditional [3H]-thymidine incorporation.
- The study elucidates the specific lymphocyte subsets involved in mitogen responses in pigs, offering valuable insights for immunology and veterinary research.
Abstract:
Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently 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 multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [3H]-thymidine incorporation data alone, it was observed that PBMC 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 detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4(+)CD8(+), CD4(-)CD8alpha(hi), CD4(-)CD8alpha(lo) and gammadelta TCR(+) cells. PWM stimulation induced responses by CD4(+)CD8(+), CD4CD8alpha(hi) but not by CD4(-)CD8alpha(lo) or gammadelta TCR(+) cells. Con A stimulation resulted in a sustained proliferation of CD8alpha(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). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs.
