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There are a number of flow-based assays that we can perform to provide valuable information on a patient's immune response. Using multi-color flow cytometric analysis of up to 7 colors, we can assess the imunophenotype and activation status of different cell populations. This can be combined with fluorescently labeled MHC class I multimers (tetramer or pentamers) to measure the number of CD8+ T cells that recognize a particular antigenic epitope.

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Multimers of peptide/MHC complexes tagged with fluorescent dyes are used as TCR ligands to identify T cells with particular antigenic specificity. This allows individual T cells to be identified by flow cytometry analysis. If the specific T cell is co-tagged with different fluorescent-conjugated antibodies that are specific for cell surface markers (cytokine or chemokine) the phenotype of the cell can also be determined.collapse

This assay is used to look for the responsiveness of T cells to a specific antigenic stimulation. Stimulation can be performed with mononuclear cells isolated from PBMC or whole blood. The assay is based on the principle by treating cells with an inhibitor of the secretory pathway to prevent secretion of the cytokine (e.g brefeldin A or monensin) the cytokines accumulate inside the cytoplasm and can be detected by staining the fixed and permeabilized cells. 

By combining the intracellular cytokine stain with staining for phenotypic markers (as well as tetramers) it is possible to determine the type of cells that produce the cytokine as well as the quantity of cytokine produced per cell. This assay requires larger sample volumes than the ELISPOT but is the only assay that determines simultaneously the type of cytokine produced by a single cell and the phenotype of such a cell.

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This cell-mediated cytotoxicity assay is an alternative to the standard chrominum-51 (51Cr) release assays. Target cells are labeled with the cell tracking dye CFSE and incubated with effector cells. At the end of the assay 7AAD (which binds to DNA and can only enter membrane-compromised cells) is added to measure cell death.

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We can perform ELISA's to look at a variety of cytokines, growth factors or other proteins in serum, plama or culture supernatant.

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This assay is used to detect and quantify the number of T cells that secrete a particular cytokine (e.g Interferon-γ) upon recognition of a specific antigen. T cells are cultured with antigen-presenting cells in wells coated with an antibody recognizing a particular cytokine. The coating antibody captures the secreted cytokine and a second cytokine-specific antibody that is coupled to a chromogenic substrate is used for detection. Results appear as spots, with each spot corresponding to one cytokine-secreting cell. The number of spots allows the calculation of the frequency of cytokine-secreting cells for a specific antigen. However this assay does not determine the amount of cytokine secreted. 

This assay is highly sensitive (1 cell in 100,000) and can be performed directly ex-vivo using relatively few T cells (compared with ICS).

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The luminex is capable of measuring up to 100 analytes. Commercially available kits can be used for the detection of multiple cytokines in serum, plasma or tissue culture supernatants. Kits consist of beads dyed with differing concentrations of two fluorophores to generate distinct bead sets. Each bead set is coated with a capture antibody specific for a specific cytokine. A second biotinylated antibody and streptavidin-phycoerythrin (S-PE) is used to detect the captured cytokine. The Luminex 200 analyzer is a dual laser, flow-based, sorting and detection platform. One laser detects the beads and determines which cytokine is being detected. The second laser determines the magnitude of PE-derived signal, which is proportional to the amount of cytokine bound.

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A flow cytometry based assay using a panel of monoclonal antibodies recognizing approximately 70% of known members of the TCR Vβ family. This can be combined with staining for other cell surface markers to allow Vβ repertoire analysis on T cell subsets.

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Gene expression analysis of the T cell receptor repertoire can provide useful information in evaluating the immune response in a variety of conditions. TCR spectratyping is a multiplex RT-PCR protocol used to analyze the T cell repertoire diversity by comparing the relative frequencies of different clonal length products within the CDR3 region of a particular TCR Vβ family. This technique is useful for analysis of clonal compositions in T cell populations, which changes during an immune reaction and can be dramatically altered in some disease states. This assay provides qualitative information regarding the diversity of the TCR Vβ repertoire but for quantitative information, the flow cytometric Vβ repertoire analysis needs to be used.

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