Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.
Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.
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正常兔血清对动物细胞凝集素抗性突变体的细胞毒性增加。
DOI：
10.1084/jem.160.4.1241
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Jones,C
中科院分区：
文献类型：
--
作者：
Jones,C
材料和方法培养中国仓鼠卵巢（CHO-K1）细胞突变体和杂交体的程序与先前描述的相同（7）。本研究中使用的细胞是J 1，一种人/中国仓鼠杂交体，其含有人11号染色体作为其唯一的人染色体，表达人细胞表面抗原，并且生长需要甘氨酸（8），以及两种CHO-K1营养缺陷型突变体，其分别显示出对天冬酰胺和尿苷的需求（9，10）。使用标准程序进行诱变实验，并通过分离暴露于添加到细胞培养基中的特异性凝集素后存活的那些诱变细胞来获得凝集素抗性克隆。在这些实验中使用的凝集素包括植物血凝素（PHA）（Difco Laboratory，Detroit，MI）、伴刀豆球蛋白A（Con A）、蓖麻毒素（RIC）和麦胚凝集素（WGA）（Sigma Chemical Co.，St. Louis，MO）和小扁豆（LCA）（Miles Laboratories，埃尔克哈特，IN）。正常兔血清（NRS）获自The杰克逊Laboratory，Bar Harbor，ME; Charles River Breeding Laboratories，威尔明顿，MA;科罗拉多血清公司，Denver，CO; Cedarlane Laboratories（Low-Tox Rabbit Complement），Hornby，安大略，加拿大;和Dutchland Laboratories Inc.，丹佛，宾夕法尼亚州。通过将恒定数量的细胞在递增浓度的凝集素中单细胞铺板并对6-7天后形成的集落数量进行评分来测定细胞的凝集素敏感性。根据存活曲线确定获得10%存活率所需的浓度。如前所述进行互补分析（11）。使用这些细胞上可用的营养标记物选择杂交种。分离杂交体并通过标准方法测试凝集素抗性。
Materials and Methods The procedures for culturing Chinese hamster ovary (CHO-K1) cell mutants and hybrids were the same as previously described (7). The cells used in this study were J 1, a human/Chinese hamster hybrid that contains human chromosome 11 as its only human chromosome, expresses human cell surface antigens, and requires glycine for growth (8), and two CHO-K1 auxotrophic mutants that show requirements for asparagine and uridine, respectively (9, 10). Mutagenesis experiments were carried out using standard procedures and lectin-resistant clones were obtained by isolating those mutagenized cells that survived exposure to the specific lectin that was added to the cell culture medium. Lectins used in these experiments included phytohemagglutinin (PHA)(Difco Laboratory, Detroit, MI), concanavalin A (Con A), ricin (RIC), and wheat germ agglutinin (WGA)(Sigma Chemical Co., St. Louis, MO), and lentil (LCA)(Miles Laboratories, Elkhart, IN). Normal rabbit serum (NRS) was obtained from The Jackson Laboratory, Bar Harbor, ME; Charles River Breeding Laboratories, Wilmington, MA; Colorado Serum Co., Denver, CO; Cedarlane Laboratories (Low-Tox Rabbit Complement), Hornby, Ontario, Canada; and Dutchland Laboratories Inc., Denver, PA. Cells were assayed for their lectin sensitivity by single-cell plating a constant number of cells in increasing concentrations of lectin and scoring the number of colonies formed after 6-7 d. The concentration needed to give 10% survival was determined from the survival curve. Complementation analysis was carried out as previously described (11). Hybrids were selected using the nutritional markers available on these cells. The hybrids were isolated and tested for lectin resistance by standard methods.