USE OF H-3-THYMIDINE FOR MEASUREMENT OF DNA-SYNTHESIS IN RAT-LIVER - WARNING
USE OF H-3-THYMIDINE FOR MEASUREMENT OF DNA-SYNTHESIS IN RAT-LIVER - WARNING
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DOI：
10.1016/0003-2697(72)90030-9
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发表时间：
1972-01-01
影响因子：
2.9
通讯作者：
KINGDON, HS
中科院分区：
文献类型：
--
作者：
MORLEY, CGD;KINGDON, HS
材料与方法雄性Sprague-Dawley白化大鼠取银条（300400 gm）， 0.85% NaCl冷冻。然后用剪刀把它们剁碎，用冰冷的生理盐水彻底清洗，然后通过三个不锈钢筛子（Dual Manufacturing Co., Chicago, illinois）。（尺寸为420，u， 250p和177p）(3)使用带手套的手指。细胞用冷盐水通过筛子清洗。得到的单细胞悬浮液在5000rpm转速下旋转5min，上清液被丢弃。将细胞颗粒用0.85%的冷盐水重悬，用17号皮下注射针轻轻穿刺细胞。根据Higgins和Anderson的方法，在乙醚麻醉下进行部分肝切除术（4)。h -甲基胸苷（比活性8.7 Ci/mmol）购自国际化学和核公司，Irvine, california）。胰蛋白酶、凝乳胰蛋白酶和cu-淀粉酶来源于营养生化。对于DNA合成的常规测量，将大约1 × lo6个细胞在Difco中37英寸孵育，Difco是最小的Eagle培养基（Spinner修改用于悬浮培养，编号5838），每毫升含有1000单位青霉素和1000 pg链霉素。将3h -甲基胸腺嘧啶（8 × 10G cpm）加入到悬浮液（终体积2ml）中，装在10ml聚乙烯管中。一般情况下，这些实验的潜伏期为15分钟至1小时。孵育后，细胞以10,000 rpm和2英寸转速快速旋转5分钟，在涡旋混合器上重悬2 ml 0.05 M EDTA钾（pH 7.0）（冰冷）中洗涤两次，然后检测放射性标记合并。用“酸”法或“苯酚”法（两种方法都在下面描述）。液体闪烁计数在Packard Tri-Carb型3375液体闪烁计数器上进行，使用10ml Aquasol液体（Nuclear-Chicago Corp.）。
MATERIALS AND METHODSLivers were excised from male Sprague-Dawley albino rats (300400 gm), and chilled in ice-cold 0.85% NaCl. They were then minced with scissors, washed thoroughly with ice-cold saline, and pushed through three stainless-steel sieves (Dual Manufacturing Co., Chicago, Ill., sizes 420, u, 250 p, and 177 p)(3) using gloved fingers. The cells were washed through the sieves with cold saline. The resulting single-cell suspension was spun for 5 min at 5000 rpm at 2” and the supernatant solution was discarded. The cell pellet was resuspended in cold 0.85% saline by gently working the cells through a 17-gage hypodermic needle. Partial hepatectomies were performed under ether anesthesia according to the method of Higgins and Anderson (4).“H-Methylthymidine(specific activity 8.7 Ci/mmole) was purchased from International Chemical and Nuclear Corp., Irvine, Calif.). Trypsin, chymotrypsin, and cu-amylase were obtained from Nutritional Biochemicals.For routine measurement of DNA synthesis, approximately 1 X lo6 cells were incubated at 37” in Difco, minimal Eagle’s medium (Spinner modified for suspension cultures, No. 5838) containing 1000 units of penicillin and 1000 pg of streptomycin per milliliter. 3H-Methylthymidine (8 X 10G cpm) was added to the suspension (final volume 2 ml) in 10 ml polyethylene tubes. In general, the incubation periods were from 15 min to 1 hr for these experiments. After incubation the cells were quickly spun down at 10,000 rpm and 2” for 5 min, washed twice by resuspension on a vortex mixer in 2 ml of 0.05 M potassium EDTA (pH 7.0)(ice cold), and then assayed for radioactive label incorporat. ed by either the “acid” technique or the ‘Lphenol” method (both described below). Liquid scintillation counting was performed in a Packard Tri-Carb model 3375 liquid scintillation counter using 10 ml of Aquasol fluid (Nuclear-Chicago Corp.).