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Detection, counting, and eventually cloning CD4 or CD8 T cells against any peptide encoded by a given gene and presented by any HLA molecule

Laboratory protocol


We set up a method to detect, count, and eventually clone CD4 or CD8 T cells against any peptide encoded by a given gene and presented by any HLA molecule. PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein. After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks, and all the individual microcultures are then screened for recognition of autologous EBV-B cells transduced with a retrovirus encoding the protein. Non transduced EBV-B cells are used as control stimulator cells. Activated T cells are detected by their expression of CD137.

Protocol Steps:
  • Stimulation of PBMC PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein.
  • Leave microcultures for 2 weeks After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks.
  • Screening of the microcultures All the individual microcultures are then screened for recognition of autologous EBV-B cells transduced with a retrovirus encoding the protein. Non transduced EBV-B cells are used as control stimulator cells.
  • Detection of activated T cells Activated T cells are detected by their expression of CD137.
Detection, counting, and eventually cloning CD4 or CD8 T cells against any peptide encoded by a given gene and presented by any HLA molecule Graph

created over 7 years ago (4 January 2010)    last modified over 5 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]