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Intracellular cytokine staining in this lab is perfoemd using the Cytofix/Cytoperm kit according to the manufacturer's protocol BD PharMingen. The first step in intracellular cytokine staining is treating T cells to inhibit protein export so that cytokine accumulates within the cells. Afterwards, the membrane of T cells is permebalized with detergent, followed by labeling of cytokines within the cells using fluorochrome-labeled cytokine-specific
antibodies. Cells can then be sorted by flow cytometry analysis, using a cell sorter that separates and counts labeled cells by laser and photomultiplier tube detectors, as shown in this figure with staining for CD8 and intracellular interferon gamma.
Flow cytometry analysis of IFN--secreting E7-specific CD8+
T cell precursors in mice vaccinated with various recombinant DNA vaccines.
Mice were vaccinated via gene gun with 2 µg of pcDNA3, E7, TAT/E7, E7/MTS,
AH/E7, or VP22/E7 DNA. One week later, mice were boosted with the same regimen
as the first vaccination. Splenocytes from vaccinated mice were cultured in
vitro with 1 µg/ml of E7 peptide (aa 49–57) overnight and analyzed
for both CD8 and intracellular IFN- by flow cytometry analysis. Vaccination
of mice with VP22/E7 DNA generated the greatest number of IFN-+ CD8+ double-positive
T cells compared with the other vaccination groups. The data from intracellular
cytokine staining shown here are from one representative experiment of two performed.
(Improving Vaccine
Potency Through Intercellular Spreading and Enhanced MHC Class I Presentation of Antigen)
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