The Intracellular Cytokine Staining (ICS) assay is a flow cytometry assay that is used to detect the production of cytokines. The assay investigates immune cells to detect a response to a target protein. First live cells are stimulated with peptide pools derived from the protein sequence under investigation. For a period of time during the stimulation, the cells are incubated with a secretory pathway inhibitor such as Brefeldin A to allow accumulation of cytokines within the cells. Next the cell membranes are permeabilized using a mild detergent. Then a cocktail of fluorescently labeled antibodies (the staining panel) is added to identify the presence of cytokines and cell surface proteins. After unbound antibody is washed out the cells are analyzed by flow cytometry to identify the combination of cytokines and cell surface markers for each cell detection event. A software package such as FlowJo is used to analyze cell populations present within the acquired data.