Immunophenotyping analysis of peripheral blood, splenic, and thymic lymphocytes in male and female rats

Abstract

A flow cytometry method was used to characterize lymphoid tissue-associated lymphocyte subsets in the Sprague-Dawley rat. Mononuclear leukocytes from peripheral blood, spleen, and thymus of male and female rats were labeled with a panel of fluorescently tagged monoclonal and polyclonal antibodies directed against specific cell-surface proteins. The differential expression of these marker proteins was used to phenotypically distinguish one subpopulation of lymphocytes from another when analyzed via flow cytometry. This method was used to determine the relative percentages and absolute number of total B cells (CD45RA+; sIgM+), total T cells (CD3+; pan-T+), helper T cells (CD3 + CD4+), cytotoxic/ suppressor T cells (CD3 + CD8+), and the CD4:CD8 ratio in each of these lymphoid tissues. Additionally, all subsets of differentiating T cells in the thymus (i.e., CD4 + CD8-, CD4-CD8+, CD4 + CD8+, and CD4-CD8-cells) were distinguished using dual parameter analyses. Results of this study demonstrate that 1) the selected panel of antibodies used in this study can identify all lymphocyte subsets present in blood, spleen, and thymus of the rat and 2) male and female Sprague-Dawley rats show slight, but not statistically significant, differences in the proportions of some lymphocyte subsets present in select lymphoid tissues. This flow cytometry method can be used to accurately assess the potential immunotoxic or immunomodulatory effect of xenobiotic agents as characterized by changes in the phenotypic expression patterns or alterations in the quantity of lymphocyte subpopulations in the rat.