Chen W F, Wilson A, Scollay R, Shortman K
J Immunol Methods. 1982 Aug 13;52(3):307-22. doi: 10.1016/0022-1759(82)90003-5.
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.
本文介绍了一种极限稀释微培养系统，其中几乎所有成熟T细胞以约1个细胞/孔的水平进行培养，在8 - 9天的时间内生长并扩增为平均有60,000个细胞的克隆。克隆效率为70 - 100%，因此扩增后的克隆集合代表了起始T细胞群体。所有Lyt表型的T细胞都能形成子代细胞克隆。该系统包括在平底微量滴定板中培养，以伴刀豆球蛋白A作为起始刺激物，同时加入经适当照射的脾填充细胞和可溶性T细胞生长因子的补充来源。所得克隆可如随附论文中所述筛选其细胞溶解功能。该系统可通过使用[³H]TdR摄取作为增殖克隆存在与否的读数，来测定能够扩增的T细胞水平或前体功能（PTL-p）。对阳性培养物频率的分析显示与预期的泊松分布拟合良好，没有复杂的抑制或辅助效应的证据。
