It steps, therefore, in a single assay, the successful completion of all the events required for antigen-specific cell-mediated cytotoxicity and is a convenient and versatile alternative to the classic51Cr-release assay. == Breadth of Immune Responses == The breadth of an antigen-specific T- or B-cell response to a pathogen or vaccine can be measured by mapping T- or B-cell epitopes. epitope mapping, and T-cell subsets). == Basic Methods == Intracellular cytokine staining(ICS), also known as cytokine flow cytometry, combined with antigen-specific T-cell stimulation is usually a widely applied technique that lends itself well to the multiparameter analysis of complex cellular samples; Nomura et al. resolved the use of polychromatic flow-cytometry (i.e. using more than five fluorescence colors) in a multicenter setting (3). In situations where multiple cytokines and surface markers need to be studied in parallelthe so-called immune function signatures in vaccination studiesICS is usually superior to the ELISPOT technology (which can, however, be performed at lower costs and is amenable to high throughput screening). In the multicenter studies reviewed by Nomura, standardized reagents were used, such as cryopreserved cells, antibodies, beads, and peptides. As would be expected in this Sitaxsentan sodium (TBC-11251) situation, the main sources of variation between sites were differences in instrument set-up and gating. Class I MHC tetramersentered the cytometry industry in 1996, whereas class II MHC multimers did so in 2002. Brooimans et al. (4) describe the standard assay approach for PE-conjugated tetramers carrying CMV epitopes from pp50 and pp65. Under routine conditions, it is possible to detect CMV-specific CD8+ T cells at frequencies as low as 1 cell/l. Chattopadhyay et al. (5) focused on Bmpr2 situations in which low-frequency CD8+ T cells bind to cognate antigen with low avidity. Here, discrimination between signal and noise can be improved by the use of a dump channel (i.e., the Sitaxsentan sodium (TBC-11251) use of specific stains to identify confounding events to be excluded from analysis), or the use of the same MHC multimer reagents labeled with different fluorochromes. Signal amplification can be Sitaxsentan sodium (TBC-11251) achieved by pretreatment of the cells with the protein kinase inhibitor, dasatinib, or the use of class I MHC multimer reagents that bind CD8 with enhanced affinity. Similar requirements, that is, to reduce nonspecific staining in the setting of rare event detection, apply to class II MHC tetramers as outlined by Cecconi et al. (6). Typically, antigen-specific CD4+ T cells occur in peripheral blood in frequencies much less than 0.2%. The staining efficiency of class II MHC tetramers is usually strongly correlated to the TCR affinity for its cognate peptide-class II MHC complex. This situation is usually in particular important for the ability of class II MHC tetramers to identify CD4+ T cells specific for autoantigens and cancer, which are mostly characterized by the expression of low-affinity TCR. Of note, CD4+ T-cell activation, rather than the overall quantitative display of TCR molecules around the cell membrane, determines the accessibility of the TCR to class II MHC tetramers. This accessibility further relies on active cell metabolism and membrane trafficking, as indicated by the requirement for a heat of 37C to achieve optimal staining results. == Live Cell Assays == Dye dilution-based proliferationassays [Wallace et al. (7)] have proven extremely useful because of their ability to correlate a critical functional outcome (i.e., growth of antigen-responsive cells) with earlier events. An important feature of the dye dilution technique is usually that division-dependent changes in the expression of cell surface markers, intracellular proteins, antigen binding, or other properties of interest can be readily quantified by flow cytometry. Among these dyes, the so-called general protein labels are reactive compounds that form random covalent bonds with amino groups on cellular proteins. Their advantages include rapid and simple use, bright fluorescence signals, and often the ability to clearly visualize distinct generations of daughter cells. Limitations arising from dye dilution can be proliferation-dependent or -impartial. The so-called general membrane labels are lipophilic compounds that integrate stably into the plasma cell membrane without binding covalently. They are simple to.