Article: article from journal or magazin.
Limit-dilution assay and clonal expansion of all T cells capable of proliferation.
Journal of Immunological Methods
A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of the expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [3H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.
Animals, Antigens, Ly/genetics, Antilymphocyte Serum/pharmacology, Clone Cells/cytology, Clone Cells/immunology, Concanavalin A/pharmacology, Cytotoxicity Tests, Immunologic/methods, Interleukin-2/biosynthesis, Kinetics, Lymphocyte Activation/radiation effects, Mice, Spleen/cytology, Spleen/immunology, T-Lymphocytes/classification, T-Lymphocytes/cytology
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