Flow cytometric analysis of intracellular interferon-gamma synthesis in rat CD4 T cells

To date the techniques used to analyse cytokine expression by rat T cells do not give information about the simultaneous production of different cytokines from individual cells. Recently, a method for analysing the intracellular production of cytokines at the single cell level using flow cytometry has been developed. It is well established that the most critical requirement for successful intracellular cytokine staining is the availability of appropriate antibodies. In rat, it is possible to stain for intracellular IL-4 and IL-10 (Th2 cytokines) using the commercially available antibodies but not for Th1 cytokines. In the present work, we show that DB1, a mouse anti-rat IFN-gamma monoclonal antibody, could be used for intracytoplasmic staining of IFN-gamma producing rat CD4 T cells. The specificity of the staining was confirmed using a molar excess of unlabelled antibodies or recombinant cytokine. Finally, intracellular staining for IFN-gamma correlates with cytokine production in culture supernatant as evaluated by ELISA analysis.