Williams R M, Benacerraf B
Lab Invest. 1975 Mar;32(3):382-7.
Various numbers of mouse thymus, lymph node, or spleen cells were cultured in serum-containing medium with concentrations of 0.06 to 64 mug. per ml. of concanavalin A (Con A), using our version of the microculture technique. Maximal DNA synthetic responses were obtained with 1.0 million thymocytes (highest number used) at 4 mug. per ml. of Con A and with 0.75 million lymph node cells or 0.5 million spleen cells at 1 mug. per ml. of Con A. At optimal Con A concentration the magnitudes of response of 0.25 million thymus, compared with the same number of lymph node cells, were very similar. However, the response of lymph node cells was superior to that of thymocytes when 0.5 million initial cells were used, whereas 1.0 million thymocytes gave a better response than did the same number of lymph node cells. Regardless of the cell concentration, a comparison of responses by the three different cell types with the same cell number showed that the Con A concentration-DNA synthetic response curve was broadest for spleen cells, narrow for thymus cells, and intermediate for lymph node cells. Results in replicate experiments were highly reproducible, and the data indicated that peak DNA synthetic responses were not directly proportional to cell number when certain numbers of cells were used; the actual proportionality of response to cell number varied with the cell type and the mitogen concentration. When examined throughout the culture period, concentrations of Con A which were above the optimal for stimulation of DNA synthesis resulted in significant decreases in the number of viable cells. Time course experiments with Con A (or phytohemagglutinin)-stimulated BALB/c spleen cells showed that the initiation of DNA synthesis occurred between 12 and 24 hours of culture, with peak levels of 3H-thymidine incorporation occurring during the 2nd and 3rd days of culture. The presence or absence of fetal calf serum had essentially no effect on the Con A- or phytohemagglutinin-stimulated DNA synthetic responses during the 3rd day of culture. These results demonstrate that detailed evaluation of culture variables and mitogen concentrations is required for the most meaningful use of mitogen-stimulated DNA synthesis as a parameter for comparing different lymphoid cell populations.
采用我们改良的微量培养技术，将不同数量的小鼠胸腺、淋巴结或脾细胞，接种于含不同浓度（0.06至64微克/毫升）伴刀豆球蛋白A（Con A）的含血清培养基中培养。每毫升含4微克Con A时，100万个胸腺细胞（所用的最高细胞数）可获得最大的DNA合成反应；每毫升含1微克Con A时，75万个淋巴结细胞或50万个脾细胞可获得最大的DNA合成反应。在最佳Con A浓度下，25万个胸腺细胞与相同数量的淋巴结细胞相比，反应强度非常相似。然而，当接种50万个初始细胞时，淋巴结细胞的反应优于胸腺细胞；而接种100万个胸腺细胞时，其反应比相同数量的淋巴结细胞更好。无论细胞浓度如何，对相同数量的三种不同细胞类型的反应进行比较发现，Con A浓度-DNA合成反应曲线以脾细胞最宽，胸腺细胞最窄，淋巴结细胞居中。重复实验结果具有高度可重复性，数据表明，当接种特定数量的细胞时，DNA合成反应峰值与细胞数量并非直接成正比；反应与细胞数量的实际比例关系因细胞类型和促有丝分裂原浓度而异。在整个培养期间进行检测时，高于刺激DNA合成最佳浓度的Con A会导致活细胞数量显著减少。用Con A（或植物血凝素）刺激BALB/c脾细胞的时间进程实验表明，DNA合成起始于培养12至24小时之间，3H-胸腺嘧啶核苷掺入的峰值出现在培养的第2天和第3天。在培养第3天，胎牛血清的存在与否对Con A或植物血凝素刺激的DNA合成反应基本没有影响。这些结果表明，为了最有效地将促有丝分裂原刺激的DNA合成作为比较不同淋巴细胞群体的参数，需要对培养变量和促有丝分裂原浓度进行详细评估。
