Dual-color flow cytometric analysis of phenotype, activation marker expression, and proliferation of mitogen-stimulated bovine lymphocyte subsets

Bovine peripheral blood mononuclear cells were cultured in vitro for 3 days with the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohemagglutinin (PHA), and anti-CD3 monoclonal antibody. Activation of T-lymphocyte subsets (CD4+, CD8+, and γδ T-cell receptor+) and of B-cells was measured by two-color flow cytometric analysis of subset expression of IL-2 receptor alpha (CD25) and MHC class II. Proliferation of lymphocyte subsets was directly measured by two-color flow cytometric analysis of fluorescence intensity of PKH2, a fluorescent dye that stably incorporates into cell membranes. CD4+ and CD8+ T-cell subsets were stimulated by all the mitogens to increase expression of IL2r and MHC II and to proliferate. δγ+ T-cells responded to all four mitogens with increased IL2r and MHC II expression. Con A and PHA caused measurable proliferation of γδ+ T-cells, but PWM and anti-CD3 did not. B-cells generally responded to the mitogens with increased IL2r and MHC II expression. B-cells proliferated when incubated with Con A, but did not measurably proliferate in response to PWM, PHA, or anti-CD3.