用于实时细胞分析仪的阵列金电极再生
1School of Public Health,Nanjing Medical University, 2School of Pharmacy,Nanjing Medical University, 3State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering,Nanjing Tech University, 4Department of Medical Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research,The Affiliated Cancer Hospital of Nanjing Medical University
Summary
该协议描述了一个通用的战略, 以重新生成商业阵列金电极装备的无标签的细胞分析仪, 旨在节省高运行成本 ofmicrochip 的化验。再生过程包括胰蛋白酶消化, 用乙醇和水冲洗, 和一个纺纱步骤, 使微晶片的重复使用。
Abstract
基于无标签细胞的检测有利于生物化学研究, 因为它不需要实验动物的使用。由于它能够提供更多的动态信息的细胞在生理条件比经典生化检测, 这种无标签的实时细胞分析的基础上的电阻阻抗原理是吸引更多的关注在过去十.然而, 由于测量成本相对昂贵, 因此它的实际利用可能受到限制, 在这种情况下, 用于细胞分析仪的昂贵的一次性黄金微晶片将被使用。在本议定书中, 我们制定了一个通用的战略, 以再生阵列黄金电极装备为一个商业无标签细胞分析仪。再生过程包括胰蛋白酶消化, 用乙醇和水冲洗, 以及纺纱步骤。该方法已经过测试, 证明对商业电子板的再生和重复使用至少三次是有效的, 这将有助于研究人员节省实时细胞化验的高运行成本。
Introduction
由于其高效和少劳动密集型的实验过程, 在过去十年中, 基于无标签的细胞技术在分析和筛选等方面取得了迅速的增长, 例如蛋白质组学1,2, 药物传递3,等等4,5与传统的生化方法相比, 针对细胞分析, 无标签的实时细胞检测与原型开发的 Giaever 和同事以前6是基于在细胞附着的微晶片表面记录电信号变化的原理, 允许以定量的方式连续测量细胞生长或迁移。根据这一策略, 采用基于电阻阻抗的检测原理的实时细胞电子传感 (RT) 系统介绍了7、8以及最近推出的商用实时单元分析仪 (RTCA)。实验室研究9。
商用实时细胞分析仪主要是从细胞增殖、迁移、生存能力、形态学、黏附力等方面对培养细胞的生理变化进行分析, 以观察其产生的电阻抗的诱发信号。微芯片的表面10,11。这种电信号由分析仪进一步转换成一个无量纲参数, 称为细胞指数 (CI) 来评估细胞的状态。微晶片阻抗的变化主要反映了电极/溶液界面上覆盖细胞的局部离子环境。因此, 细胞分析仪的分析性能很大程度上依赖于核心传感单元, 即一次性微晶片 (即, 所谓的电子板,例如, 96/16/8 井), 这是由阵列金电极lithographically 印在孵化井的底部。金电极装配在一个循环的在线格式 (图 1) 和覆盖的孵化井的大部分表面积, 这允许动态和敏感检测连接的细胞3,12,13 ,14。该词将增加, 在更多的表面覆盖细胞的芯片, 并减少当细胞暴露在毒物导致细胞凋亡。虽然实时细胞分析仪经常用于确定细胞毒性11和神经毒性15 , 并提供比经典端点方法更多的动力学信息, 但一次性电子板是最昂贵的消费品.
到目前为止, 还没有可用的方法来再生电子板, 这可能是由于诸如食人鱼溶液或乙酸等严酷的再生条件涉及16,17,18, 这可能会改变金微晶片的电状态。因此, 在电子板再生过程中, 采用温和有效的方法从金片表面去除粘附细胞和其他物质是可取的。我们最近制定了一项议定书, 目的是利用非腐蚀性试剂再生一次性电子板, 再生芯片的特点是电化学和光学方法19。利用速效和适度的实验试剂, 包括胰蛋白酶和乙醇, 建立了无不良影响的商用电子板再生的一般方法, 成功地应用于再生两种主要类型用于 RTCA 的电子板 (16 和 L8)。
Protocol
注: 一般情况下, 再生过程包括胰蛋白酶消化和漂洗步骤与乙醇和水。消化时间根据所使用的细胞数而变化, 所用细胞的类型和数量可能因实验目的而异。建议使用光学和电化学方法检查再生微晶片, 以优化再生条件。在实验中, 可溶性和不溶性的化学物质可能涉及, 这两种典型的再生程序的例子是详细的。 1. 再生溶液的制备 注: 在无菌条件下准备并处理所有…
Representative Results
金微芯片的表面特性: 在图 1中概述了此协议中使用的再生过程。 图 2显示了光学显微镜下的新鲜和再生电子版的显微表面图片。如图 2A和图 2C所示, 显微观察表明, 在新鲜和经过处理的电子板之间, 微晶片表面的光学特性几乎没有差别。使用阿霉素自发荧光?…
Discussion
我们总结了几种可用的方法17,23,24,25,26 , 用于在表 1中再生微晶片。基本上, 这些方法涉及相对苛刻的实验条件, 以实现芯片的完全再生, 因为存在着强大的分子-分子相互作用, 如用于 SPR 芯片的免疫复合体。然而, 这些苛刻的实验条件可能对 RTCA 中使用的阵列金微?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金 (U1703118) 的支持, 该项目由江苏省高等教育机构 (PAPD) 的优先学术项目开发资助, 江苏 Shuangchuang 项目, 国家重点开放式基金。化学/生物传感和化学计量学实验室 (2016015) 和大分子 (2017kf05) 国家实验室和江苏省特别指定的教授项目, 中国。
Materials
| Rat: Sprague-Dawley | Charles River | Cat# 400 | |
| mouse anti-rat CD11b monoclonal (clone OX42) | Bio-Rad | Cat# MCA275R | Panning: 1:1,000; Staining: 1:500 |
| Goat polycolonal anti-Iba1 | Abcam | Cat# AB5076 | Staining: 1:500 |
| Rabbit polyclonal anti-Ki67 | Abcam | Cat# AB15580 | Staining: 1:500 |
| Alexa Fluor Donkey anti-mouse 594 | Invitrogen | Cat# 11055 | Staining: 1:500 |
| Alexa Fluor Donkey anti-goat 488 | Invitrogen | Cat# A-21203 | Staining: 1:500 |
| Alexa Fluor Donkey anti-Rabbit 647 | Invitrogen | Cat# A-31573 | Staining: 1:500 |
| Triton-X (detergent in ICC staining) | Thermo Fisher | Cat# 28313 | |
| Heparan sulfate | Galen Laboratory Supplies | Cat# GAG-HS01 | |
| Heparin | Sigma | Cat# M3149 | |
| Peptone from milk solids | Sigma | Cat# P6838 | |
| TGF-β2 | Peprotech | Cat# 100-35B | |
| Murine IL-34 | R&D Systems | Cat# 5195-ML/CF | |
| Ovine wool cholesterol | Avanti Polar Lipids | Cat# 700000P | |
| Collagen IV | Corning | Cat# 354233 | |
| Oleic acid | Cayman Chemicals | Cat# 90260 | |
| 11(Z)Eicosadienoic (Gondoic) Acid | Cayman Chemicals | Cat# 20606 | |
| Calcein AM dye | Invitrogen | Cat# C3100MP | |
| Ethidium homodimer-1 | Invitrogen | Cat# E1169 | |
| DNaseI | Worthington | Cat# DPRFS | |
| Percoll PLUS | GE Healthcare | Cat# 17-5445-02 | |
| Trypsin | Sigma | Cat# T9935 | |
| DMEM/F12 | Gibco | Cat# 21041-02 | |
| Penicillin/ Streptomycin | Gibco | Cat# 15140-122 | |
| Glutamine | Gibco | Cat# 25030-081 | |
| N-acetyl cysteine | Sigma | Cat# A9165 | |
| Insulin | Sigma | Cat# 16634 | |
| Apo-transferrin | Sigma | Cat# T1147 | |
| Sodium selenite | Sigma | Cat# S-5261 | |
| DMEM (high glucose) | Gibco | Cat# 11960-044 | |
| Dapi Fluoromount-G | Southern Biotech | Cat# 0100-20 | |
| Poly-D-Lysine | Sigma | Cat# A-003-E | |
| Primaria Plates | VWR | Cat# 62406-456 | |
| Stock reagents | reconstitution | Concentration used | Storage |
| Apo-transferrin | 10 mg/mL in PBS | 1:100 | -20°C |
| N-acetyl cysteine | 5 mg/mL in H2O | 1:1,000 | -20°C |
| Sodium selenite | 2.5 mg/mL in H2O | 1:25,000 | -20°C |
| Collagen IV | 200 μg/mL in PBS | 1:100 | -80°C |
| TGF-b2 | 20 μg/mL in PBS | 1:10,000 | -20°C |
| IL-34 | 100 μg/mL in PBS | 1:1,000 | -80°C |
| Ovine wool cholesterol | 1.5 mg/mL in 100% ethanol | 1:1,000 | -20°C |
| Heparan sulfate | 1 mg/mL in H2O | 1:1,000 | -20°C |
| Oleic acid/Gondoic acid | Gondoic: 0.001 mg/mL; Oleic: 0.1 mg/mL in 100% ethanol | 1:1,000 | -20°C |
| Heparin | 50 mg/mL in PBS | 1:100 | -20°C |
| Solutions | Recipe | Comments | |
| Perfusion Buffer | 50 μg/mL heparin in DPBS++ (PBS with Ca++ and Mg+ +) | Use when ice-cold | |
| Douncing Buffer | 200 μL of 0.4% DNaseI in 50 mL of DPBS++ | Use when ice-cold | |
| Panning Buffer | 2 mg/mL of peptone from milk solids in DPBS++ | ||
| Microglia Growth Medium (MGM) | DMEM/F12 containing 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 5 μg/mL N-acetyl cysteine, 5 μg/mL insulin, 100 μg/mL apo-transferrin, and 100 ng/mL sodium selenite | Use ice-cold MGM to pan microglia off of immnopanning dish. | |
| Collagen IV Coating | MGM containing 2 μg/mL collagen IV | ||
| Myelin Seperation Buffer | 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 μL 1 M CaCl2, 5 μL 1 M MgCl2 | Mix well after the addition of CaCl2 and MgCl2 | |
| TGF-b2/IL-34/Cholesterol containing growth media (TIC) | MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 μg/mL ovine wool cholesterol, 10 μg/mL heparan sulfate, 0.1 μg/ml oleic acid, and 0.001 μg/ml gondoic acid | Make sure to add cholesterol to media warmed to 37 °C and do not add more than 1.5 μg/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH. |
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Citar este artículo
Xu, Z., Song, Y., Jiang, H., Kong, Y., Li, X., Chen, J., Wu, Y. Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer. J. Vis. Exp. (133), e56250, doi:10.3791/56250 (2018).