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Flow Cytometry for Phenotypic Analsys (1-7 Colors)
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.
Tetramer Staining
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.
Intracellular Cytokine Staining (ICS)

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.

Flow Cytometric-based Cytotoxicity Assays
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.
We can perform ELISA's to look at a variety of cytokines, growth factors or other proteins in serum, plama or culture supernatant.
Multiplex Cytokine Analysis

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.

TCR Vβ Repertoire Analysis (Flow Cytometry)

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.