Oreshkin E N
Vopr Virusol. 1982 Mar-Apr;27(2):235-8.
The following conditions were found to be necessary to achieve infection of a Spodoptera frugiperda cell line with purified DNA of nuclear polyhedrosis virus of Spodoptera exempta (NPV): (1) cell monolayers shall be prepared one day before the experiment; the number of cells added to 35-mm wells of plastic clusters (Falcon) shall be 1.0 X10(6)-1.1X10(6), and the cells shall be in the stage of logarythmic growth; DNA shall be diluted in HeBS-buffer (pH 6.95-7.05); the volume of the mixtures added per well shall be 0.3 to 0.5 ml; (2) DNA calcium phosphate mixture shall be prepared for 20-30 min at 20 degrees C; (3) the cells shall be inoculated with the DNA-calcium-phosphate mixture for 45-60 min at 28 degrees C; (4) dimethylsulphoxide (DMSO) shall be added 4 hours post-inoculation for 4 min; (5) the upper agarose layer (0.75% agarose) shall be added after 2 hours of incubation in the cell growth medium TC-100 after DMSO had been added. Under these conditions the specific activity of Spodoptera exempta NPHV DNA may achieve the value of 2.26 X 10(3) plaque-forming centres per 1 microgram.
发现要用纯化的草地贪夜蛾核型多角体病毒（NPV）的DNA感染草地贪夜蛾细胞系，需满足以下条件：（1）实验前一天制备细胞单层；添加到塑料培养板（Falcon）35毫米孔中的细胞数量应为1.0×10⁶ - 1.1×10⁶个，且细胞应处于对数生长期；DNA应在HeBS缓冲液（pH 6.95 - 7.05）中稀释；每孔添加的混合物体积应为0.3至0.5毫升；（2）DNA磷酸钙混合物应在20℃下制备20 - 30分钟；（3）细胞应在28℃下用DNA - 磷酸钙混合物接种45 - 60分钟；（4）接种后4小时添加二甲亚砜（DMSO），持续4分钟；（5）添加DMSO后，在细胞生长培养基TC - 100中孵育2小时后，添加上层琼脂糖层（0.75%琼脂糖）。在这些条件下，草地贪夜蛾NPV DNA的比活性可达每1微克2.26×10³个噬斑形成中心。
