• Intracellular Cytokine Staining Protocol

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Introduction

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A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly. In vitro stimulation of cells is usually required for detection of cytokines by flow cytometry since cytokine levels are typically too low in freshly-prepared cells. Stimulation of cells with the appropriate reagent will depend on the cell type and the experimental conditions. For example, to stimulate T cells to produce IFN-gamma, TNF-alpha, IL-2, and IL-4, a combination of PMA (a PKC activator) and Ionomycin (a calcium ionophore) or anti-CD3 antibodies are used. To induce IL-6, IL-10 or TNF-alpha production by monocytes, stimulation of PBMC's with LPS is used.

Note: Optimal stimulation period for induction of a given cytokine is variable and has to be determined. For example, the best time for detection of IL-6-producing cells by human LPS-activated monocytes is 6 hours, whereas IL-10 needs at least 24 hours stimulation.

In contrast to detection of secreted cytokines by ELISA, for detection of intracellular cytokines, it is necessary to block secretion of cytokines with reagents such as Monensin or Brefeldin A during the last few hours of the stimulation. It is advised that the investigators evaluate the use and efficacy of different protein transport inhibitors in their specific assay system.

Note: Generally, the non-specific background staining of fixed and permeabilized cells is higher than surface staining; therefore, extra protein such as BSA or FCS can be added to the staining buffer. Optimal concentration of the fluorochrome-conjugated anti-cytokine antibodies has to be determined experimentally. To confirm specificity of the staining, it is common to block the directly-conjugated anti-cytokine antibodies with excess amounts of unlabeled antibody. Alternatively, recombinant cytokines can be used for blocking.

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