Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation

The assessment of intracellular cytokines at the single-cell level by how c ytometry has recently become a potent tool in many areas of cell biology an d in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simul taneous determination of lymphocytes (CD3(+) and CD4(+)) or monocytes (CD33 (+) and CD14(+)) was used for comparison of phytohemagglutinin (PHA)-and ph orbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for inter leukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin s timulation caused prominent alterations of cell morphology and membrane exp ression of CD4 and CD14. In contrast, PHA did not cause downregulation of s urface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis, Moreover, during 4 8 h of culture PHA stimulated tumor necrosis factor beta and IL-10 producti on, which was not observed when PMA-ionomycin was used. We conclude that th e use of PHA for cell activation may limit in vitro artifacts and allow mor e precise analysis of intracellular cytokine production; in various disease states.