To increase the resolution when detecting antigen-specific T cells with MHC tetramers, at the same time that improves the specificity of the assay, we recommend a two color staining with the same allele/peptide combination. Furthermore, each allele can be coded with a unique two-color combination, allowing for the detection of multiple antigen-specific T cells at the same time.
A) CD8+ T Cells previously gated on lymphocytes (FSC vs SSC) and 7-AAD negative events, the exclusion cocktail contains Alexa Fluor® 700 anti- CD4, CD19, CD14, and CD16. B) Antigen-specific CD8+ T Cells, gated as described, detected with Flex-T™ tagged with PE and APC. HLA-A*11:01 Flex-T™ is loaded with EBV peptide (IVTDFSVIK).
The bar graph shows peptide exchange of HLA-A*11:01 allele. Monomers were irradiated with UV light in the presence of positive (Pos), negative (Neg), and p21 (EBV RVRAYTYSK) peptides, or no peptide (UV only). ELISA signal was normalized using the Pos control well.