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1。猫星形胶质细胞的细胞培养和治疗与H 2 O 2 <ol><li>文化细胞系G355 - 5在75厘米的 2瓶用DMEM媒体加营养素在37 ° C和5％的CO 2，直到60％融合（〜5天）。每1-2天更换介质或需要保持一个健康的文化。 </li><li>取出介质，加2毫升0.25％胰蛋白酶。电梯45秒吹打细胞 - 在室温（RT）1分钟。 </li><li>快速传输悬挂一个15毫升的锥形管中，含有10毫升的媒体。轻轻混匀。 </li><li>离心3分钟，23℃在300 XG获得沉淀。上清液丢弃，而不会干扰颗粒。 </li><li>添加6-7毫升的新鲜媒体和悬浮细胞。 </li><li> 〜1毫升的细胞，每孔6孔板。加入2-3毫升媒体，每口井和孵化，直到80-90％融合。 </li><li>准备在媒体100μm的过氧化氢 ​​溶液（H 2 O 2）。 </li><li>从每口井的媒体和替换要么是2毫升的新鲜传媒（对照组），2毫升H 2 O 2（治疗）或DPBS 2毫升。孵育于37℃，5％的CO 2为3小时。 </li><li>删除的解决方案，并添加1毫升的胰蛋白酶解除细胞。细胞转移到1.7 ml的Eppendorf管中含有0.5毫升DPBS。这一步应该是每孔单独执行，以防止长时间的胰蛋白酶孵育细胞死亡。 </li><li>旋转300 XG为3分钟，弃上清，悬浮在1毫升的DPBS。 </li></ol> 2。染色活性氧<ol><li>以最小的光线照射，以防止漂白，染色的所有步骤应进行。 </li><li>加入1μL50毫米CCCP的阳性对照。在37℃，5％CO 2为5分钟。 </li><li>添加5μL10毫米CM - H 2 DCFDA解决的H 2 O 2组。在37℃，5％CO 2为15-30分钟。无染色添加到生理盐水样品。 </li><li> 2毫升的DPBS清洗样本，以清除残留的污点。 </li><li> 3分钟，在300 XG旋获得沉淀。 </li><li>重悬在0.5毫升的DPBS细胞。每个样品中加入1μL的PI，除盐水。 </li><li>样品转移到5毫升流式细胞仪管。 </li><li>流式细胞仪上运行。 </li></ol> 3。流式细胞仪<ol><li>运行样品上贝克曼FC500配备与488 nm的氩激光及以下带通行证（或同等学历）：525，775和620 nm，所有± 20纳米。 </li><li>准备适当的控制：未染色细胞，CM - H 2 DCFDA染色只，有价证券，双染色（阴性对照）。 </li><li>做一个协议具有以下histoplots：FS与SS，有价证券与CM - H 2 DCFDA。它也应该有以下直方图：细胞的数量与FL1，FL2，FL3，对应的污渍。 </li><li>在运行样品，检查设备，液体和废物容器。至少20分钟的热身设备。 </li><li>清洁设备，并执行适当的珠子，根据厂家的标准校准。 </li><li>选择您的协议，并运行这些示例。 </li><li>清洁设备，完成以前。 </li><li>清洁真空根据厂家的标准线。 </li><li>关闭流和清洁头/真空。 </li><li>关闭软件和计算机。 </li></ol> 4。代表性的成果： 流式细胞仪检测结果进行了分析使用FlowJo 7.6和色斑的控制客观上设置的门控。在此基础上，健康的细胞出现直方图和活性氧（氧化应激）左侧是检测一个细胞转移到右侧。图1显示了健康猫的星形胶质细胞的过氧化氢的影响的结果。从FL1的数据是用来衡量CM - H 2 DCFDA，这表明存在ROS的力度。正如预期的那样，一个金额较高的活性氧与H 2 O 2处理的样品检测。这是显示在荧光强度转向由左到右的直方图。比较健康细胞用DMEM与DPBS治疗时，并没有显着的变化ROS的量（图2）。 <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="图1"/> 图1流的健康猫的星形胶质细胞的氧化应激水平和对健康细胞的H 2 O 2的效果的流式细胞仪的结果。结果表明，H 2 O 2增加在健康细胞中的ROS水平相比，与没有治疗的细胞（DMEM） 。 <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="图2"/> 图2。流量孵育DPBS 3小时的健康细胞中检测到ROS水平的流式细胞仪结果。

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通常建议的病毒的机制诱导神经元的损伤是氧化应激1，4，7，9，13，15，16，18-22，27，31 。基本上，它是通过病毒暴露的神经胶质细胞（星形胶质细胞和小胶质细胞）释放，如氧化钾，超氧阴离子自由基，一氧化氮，和/或过氧化氢，这是有毒的神经细胞的活性氧自由基。同样，氧化应激也被作为一个主要的甲基苯丙胺神经毒性2，6，17的病理机制的建议。由于本集团在病毒和药物?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM培养液</td><td> Cellgro </td><td> 15 - 013 - CV </td><td></td></tr><tr><td>纪念维生素</td><td> Cellgro </td><td> 25 - 020 - CL </td><td> 1％（V / V） </td></tr><tr><td> L -谷氨酰胺</td><td> Hyclone公司</td><td> 17 - 605E </td><td> 1％（V / V） </td></tr><tr><td>非必需氨基酸</td><td> Hyclone公司</td><td> 25 - 025 - CL </td><td> 1％（V / V） </td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30071.03 </td><td> 20％（V / V） </td></tr><tr><td>人体必需的氨基酸</td><td> Cellgro </td><td> 25 - 030 - CL </td><td> 0.2％（V / V） </td></tr><tr><td> 500毫升真空过滤系统</td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml锥形管</td><td>鹘</td><td> 352097 </td><td></td></tr><tr><td> 75厘米2组织培养瓶</td><td>鹘</td><td> 353136 </td><td></td></tr><tr><td> 6孔细胞培养板</td><td>鹘</td><td> 353224 </td><td></td></tr><tr><td> CM - H 2 DCFDA </td><td> Invitrogen公司</td><td> C6827 </td><td></td></tr><tr><td>碘化丙啶（PI） </td><td>西格玛</td><td> P4170 - 10毫克</td><td></td></tr><tr><td>胰蛋白酶</td><td>龙沙</td><td> 17 - 160F </td><td></td></tr><tr><td> H 2 O 2 </td><td>西格玛</td><td> H6520 </td><td></td></tr><tr><td> HYQ抗生素</td><td> Hyclone公司</td><td> SV30079.01 </td><td> 0.1％（V / V） </td></tr><tr><td> G355 - 5细胞</td><td> ATCC </td><td> CRL - 2033 </td><td>正常猫科动物的大脑</td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

的病毒通常建议的机制，诱导神经元的损伤是氧化应激。星形胶质细胞有重要作用，在控制对中枢神经系统（CNS）的氧化应激。星形胶质细胞有助于保持神经元以及保护神经元的活性氧（ROS）的稳态环境。 CM - H2DCFDA是一个细胞的活性氧的存在透水的指标。 CM - H 2 DCFDA作为一个非荧光化合物进入细胞，并成为荧光细胞酯酶删除后的醋酸组，该化合物是氧化。细胞，流式细胞仪检测，发现绿色荧光细胞处于氧化状态的指示。 CM - H 2 DCFDA是由不同的ROS的大量氧化易感。这种缺乏特异性，对活性氧可以氧化CM - H 2 DCFDA，使该化合物为使用宝贵的摄政在一个发病调查的早期阶段，这种检测可以用于氧化的细胞环境到屏幕上，无论是其中的氧气自由基或活性氧相结合是负责细胞的条件。一旦它被建立，活性氧是目前CM - H 2 DCFDA氧化，然后可以进行更多的实验来确定在特定的发病过程中所涉及ROS或组合罗斯。这项研究的结果表明，与过氧化氢 ​​增加CM - H 2 DCFDA的加入荧光检测与生理盐水对照，表明这个实验是一个宝贵的测试，检测G355 - 5细胞内的氧化环境猫星形胶质细胞株。

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

一种检查方法检测细胞氧化的环境，来衡量的CM - H2DCFDA氧化。一旦氧化细胞内，CM - H2DCFDA的变化，从非荧光一​​种荧光的化合物。是用流式细胞仪检测荧光的这种变化，并表示在氧化环境中的细胞数量。

筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

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14.3K 次观看.  Western University of Health Sciences. 一种检查方法检测细胞氧化的环境，来衡量的CM - H2DCFDA氧化。一旦氧化细胞内，CM - H2DCFDA的变化，从非荧光一​​种荧光的化合物。是用流式细胞仪检测荧光的这种变化，并表示在氧化环境中的细胞数量。