Cytokine Capture Assay and Intracellular Cytokine Staining

In addition to the measurement of secreted cytokines, other methods aim to measure cell-associated cytokines, either in a cytokine capture assay (CCA) where cytokines (e.g., IFN-y, IL-4) are detected on the cell surface (Fig. 6.2) [66], or more commonly by intracellular cytokine staining (ICS), where cytokine secretion is blocked by drugs such as brefeldin A or monensin, and cytokine accumulates in the cytoplasm (Fig. 6.3) [51, 67, 68].

For both methods, fluorescence-conjugated antibodies are used to bind cytokine and fluorescing cells analyzed by flow cytometry techniques; the cells are fixed

Fig. 6.2 Schematic diagram illustrating the cytokine-capture assay (CCA). These are based on the same principle as the ELISPOT assay, but secreted cytokine is captured on the cell surface and detected with fluorescent chromogen-labeled antibodies; the "stained" T cells remain viable.

Antigen-

Antigen-

Brefeldin Ics

Fig. 6.3 Schematic diagram illustrating the intracellular staining (ICS) method. Cytokine secretion is blocked by either brefeldin A or monensin. The accumulated intracellular cytokine is detected following permea-bilization of cells to permit entry of fluorescent chromogen-labeled anti-cytokine antibodies.

Fig. 6.3 Schematic diagram illustrating the intracellular staining (ICS) method. Cytokine secretion is blocked by either brefeldin A or monensin. The accumulated intracellular cytokine is detected following permea-bilization of cells to permit entry of fluorescent chromogen-labeled anti-cytokine antibodies.

and permeabilized prior to cytokine staining in the ICS method (Figs. 6.2 and 6.3). These methods have the advantage that cells can be double-stained to identify the CD antigen phenotypes of cytokine-expressing cells. However, since in the CCA cells remain alive, cell-separation techniques such as FACS or sorting with paramagnetic beads may be applied to facilitate the purification ofantigen-specific T cells and further characterization. CCA and ICS are very sensitive, permitting the ex-vivo detection of antigen-specific T cells with frequencies as low as 0.02% of the total cells. One disadvantage compared to the ELISPOT assay is that both CCA and ICS require relatively large numbers of cells. Moreover, they are both also subject to variations in cell sampling times, reagents, re-stimulation and other experimental conditions that apply to ELISPOT; fluctuating background responses may also be problematic [69].

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