英语翻译2.8.In vitro transfection experimentChondrocytes were se

英语翻译
2.8.In vitro transfection experiment
Chondrocytes were seeded into 24-well plates at a density of 1×105cells per well in 500l of culture medium and incubated for 24 h prior to transfection.The medium was discarded,and cells were washed once with PBS of the same pH of the transfection medium.Nanoparticles containing DNA (HA/CS-plasmid nanoparticles with a CS:HA weight ratio of 4:1 were used as example) were added to the cells in DMEM containing 10% FBS without antibiotics and incubated for a period of 4h.After this process ,the nanoparticles solution was discarded,and fresh culture media,supplemented with serum and antibiotics,was added to the cells.The culture medium was changed every two days during the experiment.At different time points post transfection,EGFP-positive transfected Cells were detected using fluorescence microscope(Nikon-TE2000-U,Japan).Cells were collected and re-suspended in PBS (pH 7.4),and the transfection results were measured using a fluorescence activated cell sorting (FACS) apparatus (Calibur,BD,USA) through fluorescence channel 1 (FL1) 48 h later.Cells,which were exposed only to naked DNA or CS-plasmid nanoparticles,were analyzed as controls TM(at a concentration of 4 g/ml of pEGFP).Lipofectamine 2000 was Used as a positive control fort ransfection,and was added to DMEM without serum and antibiotics,following manufacturer procedures.Solutions were incubated for a period of 4 h,with a concentration of 0.8 g/well of pEGFP.The medium was then discarded and replaced With a complete medium containing serum and antibiotics,according to instructions.All transfection experiments were performed in triplicate.An additional transfection study was performed to investigate the potential utilization of CD44 in aiding transportation of pDNA into the cell by blocking the receptor with an excess of HA.Free HA (at concentrations 10,20,and 50 times greater than the HA content of the nanoparticles ) was added to the cells and incubated for 20 min at 37?C.After incubation,polysaccharide solutions were aspirated,and nanoparticles containing DNA in solution were introduced to the cells and incubated for 4h at 37?C and 5% CO2,as described above.The transfection efficiency was determined by fluorescent microscopy and FACS,as mentioned above.

体外转染experimentchondrocytes接种到24孔板在密度为1×105cells以及500和培养基培养24小时前转.中期被丢弃,而细胞洗涤一次公共广播相同值的转染介质.纳米粒基因（医管局/ cs-plasmid纳米粒子与政务司司长：哈重量比4 :1被用来作为例子）被添加到细胞在含10%胎牛血清无抗生素和培养时间4 h.在这个过程中,纳米粒子的解决办法是丢弃,和新鲜的文化传媒,辅以血清和抗生素,添加到细胞.该培养基改变每一天在实验过程中.在不同时间点后转染,绿色荧光蛋白阳性转染细胞检测使用荧光显微镜（nikon-te2000-u,日本）.细胞收集和重新在公共电视网（7.4）,并转染结果是衡量使用荧光激活细胞分选（仪）仪器（能力,屋宇署,美国）通过荧光1频道（运动）48小时后.细胞,这是暴露在裸D NA或cs-plasmid纳米粒子,被作为对照分析商标（在浓度为4克/毫升真）.脂质体2000是用来作为阳性对照ransfection堡,并加入无血清培养和抗生素,下列制造商的程序.解决办法是培养期为4小时,用浓度为0.8克/好的真.中被丢弃和更换一个完整的含血清和抗生素,根据指令.所有的转染实验进行了一式三份.一个额外的转染研究进行调查的潜在利用CD 44在辅助运输的质粒DNA进入细胞通过阻断受体与过量的哈.免费哈（浓度10,20,和50倍大于医管局内容纳米粒）被添加到细胞培养20分钟在37?C .孵化后,多糖的解决办法是吸气,和纳米粒子含有脱氧核糖核酸的解决方案介绍了细胞培养4 h后在37?碳5%的二氧化碳,如上所述.转染效率测定荧光显微镜和流式细胞仪,如上所述.