皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠
Summary
一键小胶质细胞生物学的研究成功是小胶质细胞免疫功能的体外从中枢神经系统组织分离过程中的保存。通过旋转振动造成的高纯度和immunofunctional细胞培养物中分离的小胶质细胞所评估的荧光成像，免疫细胞化学，ELISA和以下的小胶质细胞活化的促炎性刺激脂多糖（LPS）和Pam 3 CSK 4（PAM）。
Abstract
从中枢神经系统组织的小胶质细胞的隔离是用于研究小胶质细胞生物学体外强大的调查工具。本发明方法详述的程序用于通过机械搅拌，用旋转摇动器隔离的小胶质细胞的新生鼠皮层。这个小胶质细胞的分离方法产率，表现出在体内正常的，非病理性状况指示静态小胶质细胞的形态和功能特征的高纯度皮质小神经胶质细胞。此过程还保留了小胶质细胞的免疫表型和生化功能就证明了形态变化，NF-κB（p65蛋白）的P65亚基的核转运，和标志促炎性细胞因子的分泌，肿瘤坏死因子-α（TNF-α的诱导），经脂多糖（LPS）和Pam 3 CSK 4（PAM）的挑战。因此，本分离方法保留了两个静态和激活的免疫vated小胶质细胞，提供调查在体外条件下小胶质细胞生物学的实验方法。
Introduction
小胶质细胞，CNS实质的监视巨噬细胞，包括成年哺乳动物大脑的总细胞群的约12％。小胶质细胞来源于卵黄囊骨髓前体细胞，改变细胞密度和形态在不同的细胞结构区域的成人中枢神经系统1-5范围内。在一个健康的成年人的大脑，小胶质细胞小，分枝或极性细胞，精，动态过程。相较于周边的巨噬细胞的形态，小胶质展示，可能会出现蜂窝闲置健康的大脑静止，低调的表型，但在体内成像研究表明，小胶质细胞的过程中动态地伸出和缩回，以监测他们的微环境的方式让人想起“采样和测量“6,7。
小胶质细胞是高度和差分响应于环境和病理生理改变在大脑中，切换从监视者与效应国家通常被视为他们的休息和激活状态，分别为。这在激活开关可以通过膜结合的模式识别受体（的PRRs），如Toll-样受体（TLRs），其中病原体相关分子模式响应（的PAMP），包括细菌和病毒衍生的脂蛋白的啮合来介导，核酸，碳水化合物和8-11。除的PAMP，模式识别受体也已显示出诱导小胶质细胞活化对被称为危险/损伤相关分子模式（D-AMPS），无菌的，非致病性的分子，其代表在中枢神经系统内环境稳定的扰动，如细 胞损伤12-16。一旦接合，模式识别受体引发的细胞内信号级联放大，结果在变化中的小胶质细胞形态和基因表达，具体而言，活化的小胶质细胞适应的变形虫状的表型，转运的p65的NF-κB亚基（p65蛋白）的细胞的细胞核，并且上调了机生产线CTION和促炎细胞因子，如肿瘤坏死因子-α（TNF-α），白介素-1β（IL-1β），随着活性氧（ROS）的16-24的分泌。虽然积分在CNS中的先天免疫应答，这些分泌的分子也已发现增加神经细胞的氧化应激，从而诱发并加重神经变性疾病状态，如帕金森氏症和阿尔茨海默氏病25-29。
然而，在病理状态的小胶质细胞活化的机制尚未完全了解。因此，小胶质细胞的分离是一个强大的调查工具为这些生物过程，因为许多在小胶质细胞激活体内功能可以在培养中概括。有几种方法可用于通过Percoll梯度以下CNS组织30,31的酶消化分离的小胶质细胞，包括隔离。然而，酶消化可以改变细胞通过减少细胞表面抗原的表达32，结果在每个动物的低细胞产量比本文所描述的方法中的免疫表型。具体来说，我们报告每小狗皮质的平均收益率小胶质细胞7.5×10 5细胞，而以前报道的整个中枢神经系统隔离的方法通过Percoll梯度产量3-5×10 5细胞30,33,34。本程序规避通过隔离基于其低粘附性小胶质细胞，从而保留了小胶质细胞免疫表型和功能的使用消化酶。
在本研究中，我们描述了从新生儿杂CX3CR1-GFP（CX3CR1-GFP + / – ）衍生的混合胶质细胞培养的小胶质细胞的分离通过在旋转摇床上，先前的延长机械搅拌和C57BL / 6小鼠皮层出版法24,35。我们利用前小鼠品系的小胶质细胞易于可视化，这些小鼠表达内源性CX3CR1轨迹的控制下的GFP -单核细胞特异性启动子36-38。这种方法可以产生高纯度的小胶质细胞培养物具有保藏免疫体外 ，当与细菌脂多糖（LPS）或帕姆3 CSK 4挑战就证明了形态变化，p65蛋白的核转位，和TNF-α的分泌，TLR4和TLR1 / 2激动剂元。
Protocol
再开始此协议，在出生后几天收集新生小鼠1-3（P1-3）在无菌容器中嵌套原笼床上用品的保护和温暖。它可以快速，高效地工作，通过该协议来优化小胶质收益率是很重要的。请参阅表1为完整的试剂列表。 1。仪器仪表，文化传媒，以及菜肴的制备制备出10毫升/小狗小胶质细胞完全培养基（MCM，1个最低必需培养基Earle的[存储]补充有：1 2mM L-谷氨酰胺，1mM丙…
Representative Results
保留了小胶质细胞的体外分离过程中的免疫表型和功能是至关重要的，以便能够利用这些细胞作为调查模型胶质生物学。为了说明小胶质immunofunctionality的使用本发明方法的成功保存，我们分离皮质小神经胶质细胞从新生儿P3（CX3CR1-GFP + / -小鼠和C57BL / 6）和处理的培养物与任何LPS或帕姆。 如图1A所示 ，通过搅动小胶质细胞的分离通过旋转振动保留?…
Discussion
本程序提供了一种有效的方法皮层小胶质细胞从新生小鼠的隔离。这个过程有：1）保持小胶质细胞免疫表型和功能性两方面的益处，如通过荧光成像，免疫细胞化学和ELISA测定，以及，2）使小胶质细胞在其他神经胶质细胞（星形胶质细胞和oligodentrocytes）的存在之前，成熟隔离，这可能会在培养的神经胶质成熟期间促进重要细胞 – 细胞相互作用。重要的是，分离的细胞具有较高的生存能力和均匀?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由NIEHS的R01ES014470（KMZ）的支持。
Materials
| Glucose | Sigma | G8270 | Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 °C; shelf life: 3-6 months. Used to make MCM and MGM. |
| Sodium pyruvate 100 mM (100x) | Hyclone | SH30239.01 | Store at 4 °C. Used to make MCM and MGM. |
| Penicillin/Streptomycin 10,000 units/ml (100x) | Gibco | 15140-122 | Store at -20 °C. Used to make MCM, MGM, MM, and DM. |
| L-glutamine 200 mM (100x) | Gibco | 25030-081 | Store at -20 °C. Used to make MCM and MGM. |
| Fetal Bovine Serum (Defined) | Hyclone | SH30070.03 | Filter sterilize; store at -20 °C. Used to make MCM and MGM. |
| Minimum Essential Medium Earle's (MEM) | Cellgro | 15-010-CV | Without L-glutamine. Contains Earle's salts. Used to make MCM, MGM, and DM. |
| Horse Serum | Gibco | 16050 | Filter sterilize; store at -20 °C. Used to make MCM and MGM. |
| Hanks' Balanced Salt Solution (HBSS) | Cellgro | 21-021-CV | Without calcium and magnesium. Store at 4 °C. Used to make MM. |
| HEPES 1 M | Gibco | 15630-031 | Store at 4 °C. Used to make MM. |
| T-75 Flask | Corning | 430641 | |
| 4',6-Diamidino-2-Phenylindole, dilactate (DAPI) | Invitrogen | D3571 | Used to stain cell nucleus. |
| Rabbit anti-Iba1 | Wako | 019-19741 | Used at 1/750 dilution for ICC staining of Iba1. |
| Rabbit anti-NFκB (p65) | Abcam | 7970 | Used at 1/1250 dilution for ICC staining for p65. |
| Alexafluor 594 Goat anti-Rabbit IgG (H+L) | Invitrogen | A11012 | Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC. |
| 10 ml Disposable Serological Pipet | Fisher Scientific | 13-678-11E | |
| 50 ml Disposable Centrifuge Tube | Fisher Scientific | 05-539-8 | |
| 15 ml Disposible Centrifuge Tube | Fisher Scientific | 05-539-12 | |
| Sterile Polystyrene Petri Dish | Fisher Scientific | 875713 | 100 mm x 15 mm |
| Scissor: Straight Metzembaum (scissor #1) | Roboz Surgical | RS-6010 | 1; 5 inch; used for removing head |
| Scissor: Vannas (scissor #2) |
Fine Science Tools | 15000-08 | 1; non-angled; 2.5mm cutting edge; used to open scalp |
| Scissor: Student Vannas (scissor #3) | Fine Science Tools | 91501-09 | 1; curved; used to mince brain tissue |
| Forcep: Dumont #7 (forcep #1) |
Fine Science Tools | 91197-00 | 2; used to secure nose and remove cortices |
| Forcep: Dumont #2 (forcep #2) |
Fine Science Tools | 11223-20 | 1; used to remove scalp |
| Forcep: Dumont #3 (forcep #3) |
Fine Science Tools | 11231-30 | 1; used to remove skull |
| Forcep: Dumont #5a (forcep #4) |
Fine Science Tools | 11253-21 | 1; used to remove meninges |
| Table of specific equipment | |||
| Name of Equipment | Name of Company | Catalogue Number | Comments |
| Zoom Stereo Dissection Microscope | Olympus | SZ4060 | Microscope is placed inside Laminar-Horizontal Flow Cabinet |
| Laminar-Horizontal Flow Cabinet | Nuaire | NU-201-330 | |
| Biological Safety Cabinet | Labconco | 3440001 | Class II |
| Water-Jacketed CO2 Incubator | VWR | 97025-836 | Set to 37 °C, 5% CO2 |
| Swing-out buckets | Fisher Scientific | 75006441 | To be used with Swing-out rotor |
| Swing-out Rotor | Fisher Scientific | 75006445 | Max Radius: 19.2 (cm) |
| Sorvall Legend RT+ Centrifuge (clinical centrifuge) |
Fisher Scientific | 75-004-377 | With swing-out rotor |
| AccuSpin Micro 17 microcentrifuge (tabletop microcentrifuge) |
Fisher Scientific | S98645 | With microliter rotor (24 x 1.5/2.0 ml; Cat #: 75003524) |
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Citer Cet Article
Daniele, S. G., Edwards, A. A., Maguire-Zeiss, K. A. Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates. J. Vis. Exp. (83), e51005, doi:10.3791/51005 (2014).