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Nat Protoc. 2012 Apr 12;7(5):891-902. doi: 10.1038/nprot.2012.037.

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers.

Author information

1
Center for Cancer Immune Therapy, Department of Hematology, University Hospital Herlev, Denmark.

Abstract

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.

PMID:
22498709
DOI:
10.1038/nprot.2012.037
[Indexed for MEDLINE]

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