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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.
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Stimulation of PBMC
PBMC are stimulated in limiting dilution conditions with a pool of overlapping peptides (15 amino acids) covering the protein.
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Leave microcultures for 2 weeks
After 2 rounds of in vitro stimulation, the microcultures are left without stimulation for 2 weeks.
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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.
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Detection of activated T cells
Activated T cells are detected by their expression of CD137.
created over 14 years ago
(4 January 2010)
last modified over 13 years ago
(28 September 2011)
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