C2C12 Myotubes 测量反式等离子体膜电子传输
Summary
本协议的目标是通过细胞外电子受体 spectrophotometrically 监测反式等离子体膜电子传输, 并分析这些细胞外电子受体可能发生的酶相互作用。
Abstract
反式等离子体膜电子传输 (tPMET) 在细胞内的还原应力保护和保护免受细胞外氧化剂的破坏中起着重要作用。这种从胞内还原剂向细胞外氧化剂输送电子的过程没有很好的定义。在这里, 我们提出了 C2C12 myotubes 的分光光度检测, 以监测 tPMET 利用细胞外电子受体: 水溶性四氮唑 salt-1 (WST-1) 和 26-dichlorophenolindophenol (DPIP 或 DCIP)。通过减少这些电子受体, 我们能够在实时分析中监控这个过程。随着抗坏血酸氧化酶 (AO) 和超氧化物歧化酶 (SOD) 的加入, 我们可以确定 tPMET 的哪一部分是由抗坏血酸出口或超氧化物产生的。当 WST-1 在低背景下产生稳定结果的同时, 在添加 AO 和 SOD 后, DPIP 能够重新氧化, 并通过分光光度分析证明。该方法显示了一种实时、多井、快速分光光度法, 其优点优于其它用于监测 tPMET 的方法, 如氰化钾 (FeCN) 和 ferricytochrome c 还原法。
Introduction
纯化的等离子膜降低电子受体的能力使人认为等离子膜具有固有的氧化还原容量1。以前在真菌、植物和动物中看到过, tPMET 是多个有机体共有的过程2,3,4,5。具体地说, 这一过程已在酿酒酵母、胡萝卜细胞、红细胞、淋巴细胞、骨肉瘤、黑色素瘤、巨噬细胞、骨骼肌和中性粒细胞2、3、4,5,6,7. 在通过等离子膜传输电子以减少细胞外氧化剂的过程中, tPMET 涉及许多细胞功能, 包括: 细胞生长5,8, 细胞生存能力9, 铁新陈代谢10, 细胞信号11,12,13, 和保护从活性氧种类12,14,15。由于 tPMET 在许多细胞功能中的参与, tPMET 的失衡已经被推测为某些严重的健康状况的发展做出贡献, 包括癌症16、心血管疾病17和新陈代谢综合症18。
有多种方法来监测电子在等离子膜上的传输, 但最广泛使用的技术是通过比色测定来评估细胞外电子受体的减少。通常使用的细胞外电子受体是四氮唑盐、DPIP、FeCN 和 ferricytochromec19, 20.最常用的四氮唑盐是第二代盐, 称为WST-119。与第一代四氮唑盐相比, 这种化合物在比色测定中更容易使用, 因为两个磺酸基团增加了其水溶性21。WST-1 与中间电子受体 1-甲氧基吩 methosulfate (mPMS) 相结合, 在两个单电子转移事件中减少。此还原将 WST-1 的弱色氧化形式更改为更强烈的黄色 formazan20,22。WST-1 有一个高摩尔消光系数 37 x 103 M-1cm-1, 导致高检测灵敏度21,22。DPIP 也被用作细胞外电子受体来监测 tPMET。已表明, DPIP 可以减少 extracellularly 由 tPMET 不支持中间电子受体23,24。由于缺少中间电子受体, DPIP 可以直接从等离子膜中提取电子, 而不像 WST-124。类似于 DPIP, FeCN 已被证明是减少 extracellularly 盐 tPMET 不支持中间电子受体19,24。与 WST-1 和 DPIP 不同, FeCN 具有低摩尔消光系数, 导致检测灵敏度降低9。另一个常用的细胞外电子受体监测 tPMET 是 ferricytochrome c. 类似 WST-1, ferricytochrome c 减少增加使用中间电子受体, mPMS22。与 WST-1 不同的是, ferricytochrome c 方法由于高背景和低摩尔消光系数22而不太敏感。
本文提出了一种通过分光光度法进行 tPMET 的实时分析方法。该方法利用细胞外电子受体 WST-1 和 DPIP, 因为它们都具有较高的摩尔消光系数, 而与其他常用的细胞外电子受体如 ferricytochrome c 相比, 它们的成本更低。我们使用吩 methosulfate (pms), 而不是 mPMS 他们有类似的化学成分和 PMS 是远远低于昂贵。mPMS 是光化学法稳定, 这是一个重要的特点, 商业套件, 需要长期的货架期。然而, 我们使 PMS 新的每一个化验, 所以稳定性不应该是一个问题。我们还提出一种方法来评估可能的酶相互作用 (参见图 1) 之间的细胞外电子受体和酶, 可用于进一步表征的过程 tPMET。具体地说, 酶的 AO 和 SOD 可以用来确定哪一部分的 tPMET 是由于抗坏血酸转运或胞外超氧化物释放, 两种常用的方法, 电子运输横跨等离子膜。
Protocol
Representative Results
Discussion
我们提出了两种方法, 利用细胞外电子受体, WST-1 和 DPIP, 分光光度法检测 tPMET C2C12 myotubes。随着标准培养过程中细胞系的生长和分光光度计板读取器的增加, 在简单的微板块实验中, 可以用这些电子受体监测 tPMET。WST-1 的还原是从良好到良好的在一个试验中重现, 但有日常的变化。以 PBS 为缓冲器的日日变化系数 (cv) 为 0.18, 以哈佛商学院为缓冲区的 cv 为0.23。
从以前的文献使用 …
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢托马斯贝尔, 林恩 Mattathil, 马克 Mannino, 和 Neej 的技术支持。这项工作得到美国公共卫生服务奖 R15DK102122 从国家糖尿病和消化和肾脏疾病研究所 (NIDDK) 的支持乔纳森费舍尔。手稿内容完全是作者的责任, 不一定代表 NIDDK 或国立卫生研究院的官方意见。
Materials
| C2C12 myoblasts | American Type Culture Collection | CRL-1772 | |
| Dulbecco's modified eagle's medium – low glucose | Sigma | D6046 | |
| Fetal Plex animal serum complex | Gemini Bio-Products | 100-602 | |
| penicillin-streptomycin | Sigma | 516106 | |
| horse serum | Gibco Technologies | 16050-130 | |
| Dulbecco's phosphate buffered saline | Sigma | D8537 | |
| trypsin-EDTA | Sigma | T4049 | |
| 15 cm culture dishes | TPP | 93150 | |
| 96 well culture plates | TPP | 92096 | |
| 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1) | Accela ChemBio Inc | SY016315 | |
| phenazine methosulfate | Sigma | P9625 | |
| L-ascorbic acid | Sigma | A5960 | |
| ascorbate oxidase | Sigma | A0157 | |
| superoxide dismutase | Sigma | S5395 | |
| 2,6-dichloroindophenol sodium salt | ICN Biomedicals | 215011825 | |
| D-(+)-glucose | Sigma | G7528 | |
| HEPES sodium salt | Sigma | H3784 | |
| sodium chloride | Sigma | S7653 | |
| potassium chloride | Fisher Scientific | BP366 | |
| magnesium sulfate heptahydrate | Sigma | M5921 | |
| calcium chloride dihydrate | Sigma | C7902 | |
| potassium phosphate | Fisher Scientific | BP363 | |
| Pierce BCA Protein Assay Kit | Thermo Scientific | 23225 | |
| Powerwave X-I spectrophotometer | Biotek Insturments | discontinued | |
| Spectronic Genesys 5 Spectrophotometer | Thermo Scientific | 336001 | |
| PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile | MidSci | 781602 | |
| Iron (II) chloride tetrahydrate | Sigma | 220299 | |
| Iron (II) sulfate heptahydrate | Sigma | 215422 | |
| hypoxanthine | Sigma | H9636 | |
| xanthine oxidase | Sigma | X4500 | |
| Excel | Microsoft | ||
| R Studio | Rstudio | https://www.rstudio.com/products/rstudio/ | |
| KC4 | Biotek Insturments | discontinued |
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