Determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture.

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-gamma and IL-4 within the cell to detectable levels.