Culture and propagation of Hprt mutant T-lymphocytes isolated from mouse spleen
Culture and propagation of Hprt mutant T-lymphocytes isolated from mouse spleen
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DOI：
10.1002/(sici)1098-2280(1998)32:3
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发表时间：
1998-01-01
影响因子：
2.8
通讯作者：
Walker, VE
中科院分区：
文献类型：
--
作者：
Meng, QX;Skopek, TR;Walker, VE
小鼠t细胞在体外相对较差的生长潜力阻碍了小鼠淋巴细胞Hprt突变检测的优化，这导致克隆效率低，并且限制了Hprt突变克隆用于对照和处理小鼠突变分子分析的扩增。在本研究中，通过添加和操作豆蛋白A （Con A）、小鼠白细胞介素-2 （IL-2）和市售的培养补剂——含Con A的大鼠T-STIM(TM)，来确定产生相对较高CEs的小鼠t细胞生长条件。在添加10%大鼠T-STIM（TM）的培养基中，加入适量的Con A进行启动，外源IL-2进行克隆，从对照小鼠（n = 32）或暴露于1,3-丁二烯的小鼠（n = 27）脾脏分离的淋巴细胞中平均ce为15-16%。此外，还评估了几种试剂刺激Hprt突变克隆长期生长的潜力；这些t细胞刺激剂包括Con A、植物血凝素和钙离子载体离子霉素与肿瘤促进剂12-肉豆蔻酸13-醋酸酯结合。在一项初步研究中，用Con a刺激被证明是在各种测试条件下繁殖小鼠t细胞克隆的最有效手段。在后续实验中，将突变克隆转移到24孔板上，并在IL-2和大鼠T-STIM（TM）补充培养基中反复使用Con A刺激，发现536个突变克隆中76%的克隆扩增到每个克隆约40万至数百万个细胞。这些数据表明，添加大鼠T-STIM（TM）的培养基增强了小鼠t细胞的初始生长，并且在IL-2和大鼠T-STIM（TM）存在的情况下，用Con A重复刺激有丝分裂为繁殖小鼠t细胞克隆提供了一种手段，可以通过多种方法进行突变分析。环绕。生物化学学报，1998,32(3):344 - 344。
The optimization of the mouse lymphocyte Hprt mutation assay has been impeded by the relatively poor growth potential of mouse T-cells in vitro, which leads to low cloning efficiencies (CEs) and limited expansion of Hprt mutant clones for molecular analysis of mutations occurring in control and treated mice. In this study, the addition and manipulation of concanavalin A (Con A), mouse interleukin-2 (IL-2), and a commercially available culture supplement, rat T-STIM(TM) with Con A, were used to identify growth conditions producing relatively high CEs for mouse T-cells. Supplementation of medium with 10% rat T-STIM(TM), along with appropriate amounts of Con A for priming and exogenous IL-2 for cloning, resulted in average CEs of 15-16% in lymphocytes isolated From spleens of control mice (n = 32) or mice exposed to 1,3-butadiene (n = 27). In addition, several reagents were assessed for their potential to stimulate long-term growth of Hprt mutant clones; these T-cell stimulatory agents included Con A, phytohemagglutinin, and a calcium ionophore ionomycin combined with a tumor promoter phorbol 12-myristate 13-acetate. In a pilot study, stimulation with Con A proved to be the most effective means for propagating mouse T-cell clones under the various conditions tested. In follow-up experiments, transfer of mutant clones to 24-well plates and repeated stimulation with Con A in IL-2 and rat T-STIM(TM) supplemented medium was found to expand 76% of 536 mutant clones to about 400,000 to several million cells per clone. These data indicate that rat T-STIM(TM)-supplemented medium enhances the initial outgrowth of mouse T-cells, and that repeated mitogenic stimulation with Con A in the presence of IL-2 and rat T-STIM(TM) provides a means For propagating mouse T-cell clones for mutation analyses by a variety of methods. Environ. Mol. Mutagen. 32:236-243, 1998 (C) 1998 Wiley-Liss, Inc.