组织培养

The second passage was obtained after the cell overgrew the bottom of the culture flask and purified by trypsinization and adherence repeatedly methods.

方法1。用组织块贴壁培养法获得原代卵巢癌CAFs和卵巢NFs，细胞铺满培养瓶后首次传代，通过胰酶消化法和反复传代法进行细胞的纯化；2。

Methods The mouse uterus of 1-3 days implantation embryos were separated, sheared and cultivated. Inner cell masses were dissociated and passaged in two days according to routine method of ES cells digesting and passaging.

分离小鼠胚胎着床初期1～3天（孕4.5～6.5天）小鼠子宫，剪碎后进行组织块培养，2天内分离内细胞团，以后按常规的胚胎干细胞培养及传代方法进行传代。

Thus a practical teaching system, which is suitable for different levels and plats and throughout the whole educational process, has been established.

西南石油大学材料科学与工程学院结合石油天然气特色，以&材料的制备－组织结构－性能－应用&为主线设计专业基础实验、专业综合实验，并与企业建立&双证&教育，以培养具有较强创新和动手能力、能够满足社会需要的人才，构建了不同层次和平台、贯穿培养全过程的实践教学体系。

This investigation was to establish a simplified culture system to isolate and passage brain tumor stem cells from human medulloblastoma, observe their proliferation and differentiation, and determine the expression of normal neural stem cell antigens, CD133 and Nestin, in BTSCs. METHODS: Eleven specimens of medulloblastoma were acutely dissociated and triturated into single cells without trypsin digestion.

本研究旨在利用简化的无血清培养基和悬浮培养方法，从人髓母细胞瘤中分离培养脑肿瘤干细胞，观察其在体外的增殖、分化，鉴定其特异性标志物CD133和Nestin的表达，验证肿瘤组织切片中CD133阳性细胞的存在，为脑肿瘤干细胞的进一步研究打下基础。

METHODS: BMSCs were obtained from Japanese big-eared rabbits, and in vitro cultured. Then the subcultured BMSCs were transfected by pCDNA3.1 plasmid, followed by incubation on swine SIS to construct the tissue-engineered skin. The growth of cells and phenotype of BMSCs were detected by flow cytometry.

日本大耳兔骨髓间充质干细胞进行体外培养扩增后，通过pCDNA3.1质粒将碱性成纤维细胞生长因子基因转染至生长状态良好的骨髓间充质干细胞，并将转染后的细胞接种于制备好的猪小肠黏膜下层上，进行体外联合培养，构建组织工程皮肤。

Methods: 1 SMCs are isolated from canine blood vessels by tissue plant method.

1采用组织贴块法培养犬的血管平滑肌细胞，并进行细胞传代培养。

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

This research used Hepato-pancreas of Ctenopharyngodon idellus as experimental material to study toxicological effect of olaquindox on liver cell and pancreas exocrine cell of Ctenopharyngodon idellus, using the method of histochemisiry, primary cell culture, and immunocytochemistry, and the cells were observed by TEM and LSCM.

本研究以草鱼肝胰脏(Hepato-pancreas)为试验材料，通过原代细胞培养，组织化学，免疫荧光细胞化学等实验方法，运用透射电子显微镜，激光共聚焦扫描显微镜等手段，观察三种不同浓度的喹乙醇对体外原代培养的草鱼肝细胞和胰腺外分泌部细胞的毒理影响。

The cell biological features were observed by inverted phase contrast microscope, l ight microscope, electron microscope, cell vital ity assay, cell growth curve and cells staining after harvest and during the periods of culturing the primary, the 1st passage and 2nd passage.

分别在取材后、原代、第1 代、第2 代细胞培养期间，进行髓核细胞活力测定；爬片培养后进行甲苯胺蓝、HE、聚集蛋白聚糖番红O、Ⅰ型及Ⅱ型胶原免疫组织化学染色观察；MTT 法绘制髓核细胞生长曲线，并行原代及第2 代细胞透射电镜观察，对体外细胞的生物学特性进行研究。

METHODS: Lipofectamine method: The BMMSCs were obtained from the tibias and femurs of the guinea pigs. The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours, followed by fetal bovine medium for 48 hours. Immunohistochemistry was performed for transient expression. G418 was added after 48 hours.

①脂质体法：取体外分离、培养的第3代豚鼠骨髓间充质干细胞，将质粒-脂质体混合物加入含细胞的培养基中培养6 h，再加入胎牛血清的培养基，孵育48 h 后行免疫组织化学检测，即为瞬时表达。48 h后加入含G418培养基筛选。

They weren't aggressive, but I yelled and threw a rock in their direction to get them off the trail and away from me, just in case.

他们没有侵略性，但我大喊，并在他们的方向扔石头让他们过的线索，远离我，以防万一。

In slot 2 in your bag put wrapping paper, quantity does not matter in this case.

在你的书包里槽2把包装纸、数量无关紧要。