Meng Q, Skopek T R, Walker D M, Hurley-Leslie S, Chen T, Zimmer D M, Walker V E
Wadsworth Center, New York State Department of Health, School of Public Health, State University of New York at Albany, 12201-0509, USA.
Environ Mol Mutagen. 1998;32(3):236-43.
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 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, 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 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-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 provides a means for propagating mouse T-cell clones for mutation analyses by a variety of methods.
小鼠淋巴细胞次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（Hprt）突变试验的优化受到小鼠T细胞体外生长潜力相对较差的阻碍，这导致克隆效率（CEs）较低，并且用于分析对照小鼠和处理小鼠中发生的突变的Hprt突变克隆的扩增有限。在本研究中，使用刀豆蛋白A（Con A）、小鼠白细胞介素 - 2（IL - 2）以及市售培养补充剂（含Con A的大鼠T - STIM）的添加和操作来确定能为小鼠T细胞产生相对较高CEs的生长条件。用10%大鼠T - STIM补充培养基，同时添加适量的用于启动的Con A和用于克隆的外源性IL - 2，从对照小鼠（n = 32）或暴露于1,3 - 丁二烯的小鼠（n = 27）脾脏中分离的淋巴细胞的平均CEs为15 - 16%。此外，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、植物血凝素以及与肿瘤促进剂佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯联合使用的钙离子载体离子霉素。在一项初步研究中，在测试的各种条件下，Con A刺激被证明是扩增小鼠T细胞克隆的最有效方法。在后续实验中，将突变克隆转移到24孔板中，并在补充有IL - 2和大鼠T - STIM的培养基中用Con A重复刺激，发现536个突变克隆中的76%可扩增至每个克隆约400,000至数百万个细胞。这些数据表明，添加大鼠T - STIM的培养基可增强小鼠T细胞的初始生长，并且在IL - 2和大鼠T - STIM存在下用Con A进行反复有丝分裂刺激为通过多种方法进行突变分析而扩增小鼠T细胞克隆提供了一种手段。
