Summary

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Published: May 05, 2020
doi:

Summary

斧头迁移是运动神经元健康的重要机制。在该协议中,我们提供了一种使用微流体室跟踪运动神经元斧头酸性隔间和线粒体的斧头传输的详细方法。

Abstract

运动神经元 (MNs) 是具有长斧子的高度偏振细胞。Axonal 传输是 MN 健康的关键机制,有助于神经元的生长、发育和生存。我们描述了一种使用微流体室 (MfCs) 来跟踪 MN 斧子中荧光标记的细胞器的六角传输的详细方法。该方法速度快,价格相对便宜,便于在空间和时间中监测细胞内线索。我们描述了一个分步协议:1) 制造聚二甲基硅氧烷(PDMS)MFCs;2) 在MFC中电内脊髓外泄和MN分离培养的电镀;3) 线粒体和酸性隔间的标签,然后进行实时共聚焦想象;4) 手动和半自动六角运输分析。最后,我们证明了HB9::GFP腹腔脊髓外植性强子的线粒体和酸性隔间在运输上的差异,以证明系统的有效性。总之,该协议为研究各种六角组件的斧头传输提供了一个有效的工具,以及 MFC 使用的简化手册,以帮助发现空间实验的可能性。

Introduction

MN是具有长斧子的高度极化细胞,在成年人类中达到一米长。这种现象对维护 MN 连接和功能带来了严峻的挑战。因此,MN 依赖于信息、细胞器和材料的适当传输,沿着斧子从细胞体到突触和背部。各种细胞成分,如蛋白质、RNA和细胞器,通过斧子定期穿梭。线粒体是经常在MN中运输的重要细胞器。 线粒体对于MN的正常活动和功能至关重要,负责ATP的提供、钙缓冲和信号过程11、2。2线粒体的斧子运输是一个经过深入研究的过程33,4。4有趣的是,线粒体迁移的缺陷报告涉及几个神经退行性疾病,特别是MN疾病5。酸性隔间是沿着 MN 斧子移动的内在细胞器的另一个例子。酸性隔间包括分裂体、内体、跨Golgi仪器和某些分泌囊泡6。酸性隔间的斧头运输缺陷在一些神经退行性疾病中也被发现,最近的论文强调了它们在MN疾病中的重要性。

为了有效地研究六角运输,经常使用分离体形和体素室的微流体室9,9,10。微流体系统的两大优点,以及分块化和亚克斯的分离,使得它非常适合研究亚细胞过程11。神经元细胞体和斧子之间的空间分离可用于操纵不同神经元隔间(例如,斧子与躯体)的细胞外环境。生物化学、神经元生长/退化和免疫荧光检测都受益于这个平台。MfCs还可以通过与其他细胞类型(如骨骼肌12、13、14),13,14等神经元进行协管,帮助研究细胞与细胞之间的交流。

在这里,我们描述了一个简单而精确的协议,用于监测运动神经元中的线粒体和酸性隔间传输。通过比较逆行和逆行运动细胞器的相对百分比以及运输速度的分布,进一步展示了该方法的使用。

Protocol

本议定书对动物的护理和治疗是在特拉维夫大学动物伦理委员会的监督和批准下进行的。 1. MFC 制备 初级模具中的 PDMS 铸件 (图 1) 按照详细的协议9购买或创建原模(晶圆)。 在继续涂层步骤之前,使用加压空气清除晶圆平台的任何类型的污垢。晶圆表面应看起来光滑清晰。 向容器中注入 50 mL 液氮。准…

Representative Results

按照所述协议,小鼠胚胎HB9::GFP脊髓外植在MFC中培养(图4A)。ex植物生长了7天,当斧子完全进入远端隔间。线粒体深红色和Lysotracker红色染料被添加到远端和近端隔间,以标记线粒体和酸性隔间(图4C)。对远端凹槽中的Axons进行了成像,对电影进行了分析如下:首先,我们使用kymograph分析(?…

Discussion

在此协议中,我们描述了一个系统,用于跟踪运动神经元中线粒体和酸性舱的斧头迁移。这种简化的体外平台允许精确控制、监测和操作亚细胞神经元隔间,从而能够对运动神经元局部功能进行实验分析。该协议可用于研究MN疾病,如ALS,专注于了解在疾病10,16,16的斧路运输功能障碍的基本机制。此外,该系统还可用于研究营养因子9<…

Divulgations

The authors have nothing to disclose.

Acknowledgements

这项工作得到了以色列科学基金会(ISF,561/11)和欧洲研究理事会(ERC,309377)的资助。

Materials

35mm Fluodish – glass bottom dish World Precision Instruments WPI FD35-100
50mm Fluodish – glass bottom dish World Precision Instruments WPI FD5040-100
Andor iXon DU-897 EMCCD camera Andor
ARA-C (Cytosine β-D-arabinofuranoside) Sigma-Aldrich C1768 stock of 2mM in filtered DDW
B-27 Supplement (50X) Thermo Fisher 17504044
BDNF Alomone Labs B-250 Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Biopsy punch 1.25mm World Precision Instruments WPI 504530 For preperation of large MFC
Biopsy punch 6mm World Precision Instruments WPI 504533 For preperation of small MFC
Biopsy punch 7mm World Precision Instruments WPI 504534 For preperation of large MFC
Bitplane Imaris software – version 8.4.1 Imaris
Bovine Serum Albumine (BSA) Sigma-Aldrich #A3311-100G 5% w/v in ddw
Chlorotrimetylsilane Sigma-Aldrich #386529-100ML
CNTF Alomone Labs C-240 Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Density Gradient Medium – Optiprep Sigma-Aldrich D1556
Deoxyribonuclease I (DNAse) from bovine pancreas Sigma-Aldrich DN-25 stock 10mg/mL in neurobasal
Dow Corning High-vacuum silicone grease Sigma-Aldrich Z273554-1EA For epoxy mold preperation
DPBS 10X Thermo Fisher #14200-067 dilute 1:10 in ddw
Dumont fine forceps #55 0.05 × 0.02 mm F.S.T 1125520
Epoxy Hardener Trias Chem S.R.L IPE 743 For epoxy mold preperation
Epoxy Resin Trias Chem S.R.L RP 026UV For epoxy mold preperation
FIJI software ImageJ
GDNF Alomone Labs G-240 Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Glutamax 100X Thermo Fisher #35050-038
HB9:GFP mice strain Jackson Laboratories 005029
HBSS 10X Thermo Fisher #14185-045 Dilute 1:10 in ddw with addition of 1% P/S and filter
iQ software Andor
Iris scissors, curved, 10 cm AS Medizintechnik 11-441-10
Iris scissors, straight, 9 cm AS Medizintechnik 11-440-09
Laminin Sigma-Aldrich #L-2020
Leibovitz's L-15 Medium Thermo Fisher 11415064
LysoTracker Red Thermo Fisher L7528
Mitotracker Deep-Red FM Thermo Fisher M22426
Neurobasal medium Thermo Fisher 21103049
Nikon Eclipse Ti micorscope Nikon
Penicillin-Streptomycin (P/S) Solution Biological Industries 03-031-1
Poly-L-Ornithin (PLO) Sigma-Aldrich #P8638 Dilute 1:1000 in flitered 1X PBS
Sylgard 184 silicone elastomer kit DOW Corning Corporation #3097358-1004
Trypsin from bovine pancreas Sigma-Aldrich T1426 stock 25 mg/mL in 1XPBS
Vannas spring microdissection scissors, 3 mm blade F.S.T 15000-00
Yokogawa CSU X-1 Yokogawa

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Citer Cet Article
Altman, T., Maimon, R., Ionescu, A., Pery, T. G., Perlson, E. Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System. J. Vis. Exp. (159), e60993, doi:10.3791/60993 (2020).

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