Cytokine staining to quantify specific T cell responses in macaques optimized.

2004 SEP 1 - (NewsRx.com & NewsRx.net) -- Scientists have optimized intracellular cytokine staining for the quantitation of antigen-specific CD4+ T cell responses in rhesus macaques.

According to a study from the United States, "Standard proliferation assays used for analysis of CD4+ T cell function have significant shortcomings, including limited sensitivity, lack of truly quantitative readouts and significant variability. We have optimized an intracellular cytokine staining (ICS) assay in rhesus macaques which allows us to identify virus-specific CD4+ T cells at the single-cell level with high sensitivity while reducing background staining to a minimum. A variety of parameters were tested to determine the optimal experimental conditions necessary for the detection of antigen-specific CD4+ T cells in macaques."

"Central to our optimized protocol was the addition of cross-linked costimulatory anti-CD28 and anti-CD49d monoclonal antibodies, a modification which resulted in up to three-fold enhancement of the frequency of TNF-alpha-secreting CD4+ T cells following superantigen- or antigen-specific stimulation," said Marie Claire Gauduin at Harvard University and collaborators throughout the U.S. "The ICS protocol was also optimized with respect to antigen concentration and duration of antigerric stimulation. These modifications resulted in a convenient and highly reproducible assay with intra- and inter-assay variability of less than 10%."

"Although cryopreservation of PBMC ...