ASSOCIATION BETWEEN SPINDLE APPARATUS AND REOVIRUS
ASSOCIATION BETWEEN SPINDLE APPARATUS AND REOVIRUS
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DOI：
10.1073/pnas.50.2.268
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发表时间：
1963-01-01
影响因子：
11.1
通讯作者：
DALES, S
中科院分区：
文献类型：
--
作者：
DALES, S
材料与方法。-感染细胞、取样和病毒分析的程序：L2细胞株和呼肠孤病毒3型（Dearing株）的来源以及两者的繁殖技术已在前面描述过。37使用前，病毒裂解液至少冷冻和解冻五次，以释放细胞相关颗粒。以每个细胞4-8 PFU的输入倍数将5 × 106-107个细胞/ml与病毒悬浮液混合，并在370下连续搅拌2小时以促进快速吸附，可获得部分同步感染。经两轮洗涤和低速离心去除未吸附的病毒后，用温热的营养培养基将感染细胞稀释至2 × 106/ml浓度，悬液孵育。每隔一段时间取出样本进行病毒测定、有丝分裂指数测定和电子显微镜检查。3 .有丝分裂指数：从5 ml样品中取出约106个细胞，在800 q离心成球2分钟，然后在0.1 ml营养培养基中重悬。在显微镜载玻片上，将一液滴的细胞悬浮液与一液滴的染色液（2%醋酸或40%醋酸）混合，并用盖玻片覆盖。随后染色质的强烈染色有助于有丝分裂、间期或染色质结构异常的细胞计数。
Materials and Methods.-Procedures for infecting cells, sampling, and virus assays: The source of the strain L2 cells and of reovirus type 3 (Dearing strain) and the techniques for the propagation of both have been described previously. 3 7 Before use, virus lysates were frozen and thawed at least five tines to release cell-associated particles. Partial synchrony of infection was obtained by mixing 5 X 106-107 cells/ml with virus suspensions at an input multiplicity of4-8 PFU per cell, and by agitating the mixture continuously for 2 hr at 370 to promote rapid adsorption. After removing unadsorbed virus by two cycles of washing and low-speed centrifugation, the infected cells were diluted to a concentration of 2 X 106/ml withwarm nutrient medium and allowed to incubate in suspension. At desired intervals samples were removed for virus assays, deter-mination of the mitotic index, and electron microscopy. Concentration of infectious particles was assayed as in Gomatos et al. 3The mitotic index: Approximately 106 cells were removed in 5 ml samples, centrifuged into pellets at 800 q for 2 min, and resuspended in 0.1 ml of nutrient medium. One droplet of the thick cell suspension was mixed on a microscope slide with a droplet of stain solution (2% aceto-orcein in 40% acetic acid) and covered by a cover slip. The ensuing intense staining of chromatin facilitated the counting of cells in mitosis, in interphase, or with abnormal chromatin configurations.