筛选法测定氧化应激在猫的星形胶质细胞的细胞线，G355 - 5
Summary
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。
Abstract
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。
Protocol
1。猫星形胶质细胞的细胞培养和治疗与H 2 O 2 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。 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。 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。 离心3分钟?…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| DMEM | Cellgro | 15-013-CV | |
| MEM Vitamins | Cellgro | 25-020-cl | 1% (v/v) |
| L-glutamine | Hyclone | 17-605E | 1% (v/v) |
| Non Essential Amino Acids | Hyclone | 25-025-cl | 1% (v/v) |
| FBS | Hyclone | SH30071.03 | 20% (v/v) |
| Essential Amino Acids | Cellgro | 25-030-cl | 0.2% (v/v) |
| 500ml vacuum filtration system | VWR | 87006-076 | |
| 15 ml conical tubes | Falcon | 352097 | |
| 75 cm2 tissue culture flasks | Falcon | 353136 | |
| 6 well tissue culture plate | Falcon | 353224 | |
| CM-H2DCFDA | Invitrogen | C6827 | |
| Propidium Iodide (PI) | Sigma | P4170-10mg | |
| Trypsin | Lonza | 17-160F | |
| H2O2 | Sigma | H6520 | |
| HyQ Antibiotic | Hyclone | SV30079.01 | 0.1% (v/v) |
| G355-5 cells | ATCC | CRL-2033 | Normal feline brain |
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Citer Cet Article
Testa, M. P., Alvarado, O., Wournell, A., Lee, J., Guilford, F. T., Henriksen, S. H., Phillips, T. R. Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5. J. Vis. Exp. (53), e2841, doi:10.3791/2841 (2011).