一种方法为Target和隔离气道支配感觉神经元的小鼠
Summary
Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.
Abstract
体感神经转导热，机械，化学，和引起的内源和环境因素有害刺激。这些传入神经元的细胞体位于感觉神经节内。感觉神经节支配身体的特定器官或部分。例如，背根神经节（DRG）位于脊柱和整个身体和四肢延伸工艺。三叉神经节位于头盖骨和支配面，和上呼吸道。结状神经节的迷走神经传入整个肠道，心脏和肺延长。结状神经元控制的功能，例如一个多样化：呼吸率，气道发炎，和咳嗽反射。因此，要理解和操纵它们的功能，这是至关重要的，以鉴定和分离气道特定的神经元亚群。在小鼠中，气道暴露于荧光示踪剂染料，坚牢蓝，对特定气道结状神经元逆行示踪秒。结状神经节解离和荧光激活细胞（FAC）排序是用来收集染料阳性细胞。接着，高品质的核糖核酸（RNA）从用于下一代测序染料阳性细胞中提取。使用特定的神经元基因表达被确定这种方法的气道。
Introduction
体感神经转导热，机械，化学，和引起的内源和环境因素有害刺激。这些传入神经的细胞体存在于感觉神经节，如背根，三叉神经痛，或节状神经节。每个感觉神经节支配的身体的特定区域，并包含支配该区域内的单独的器官和组织的细胞。例如，背根神经节（DRG）位于脊柱和整个身体和四肢延伸过程，而三叉神经节位于头骨，包含支配脸，眼睛，脑膜或上气道1的神经元， 2。迷走神经的节状神经节位于颅骨下颈部和包含在整个胃肠道，心脏扩大的神经纤维细胞体，和下呼吸道和肺部3。在人类的结状神经节是独立的，但是，在小鼠它所稠合与颈神经节，这也支配肺部4。此稠合神经节通常被称为颈/结状复杂，迷走神经的神经节，或简单地结状神经节5。这里，它被称为结状神经节。
结状的传入纤维从内脏信息传递到脑干的孤束（NTS）的核心。感觉输入到这个独特神经节控制的功能，例如肠能动性6，心脏速率7，呼吸8,9，和刺激性激活呼吸道反应-10,11-一个多样化。同的功能和神经支配的器官这种多样性，这是定位和分离结状神经节的器官特异性亚群为了研究个体神经通路的关键。然而，鉴于结状的小尺寸和神经元的有限数量的包含这不是一个简单的任务。每只小鼠结状神经节中含有大约5000元12除了支持卫星细胞的广泛的人口。 5％支配气管 – 5000结状神经元中，只有3。因此，气道神经支配的神经元内由于呼吸刺激或病状的任何功能，形态或分子变化，将在密集结状神经节丢失。
为了解决这个问题，一种方法的开发是为了鉴定和分离那支配气道的神经元。呼吸道暴露于荧光示踪染料，以确定在随后支配结状神经元。耐晒蓝是由神经元拾起并迅速传播到它保留长达八周的13元胞体– 15。一旦确定，温和的，但有效的，解离的协议被用来保存染料标记和细胞生存力荧光活化的细胞（FAC）排序。分选的细胞用于提取高质量核糖核酸（RNA），以确定基因表达或f或其他下游分子分析。这个协议提供了用于分离该支配的关心组织的感觉神经元的有用和健壮的技术。
Protocol
Representative Results
Discussion
这个协议描述为目标气道神经支配的神经元中的迷走神经的节状神经节的方法。一旦标记的神经节轻轻解离最佳地保持细胞数量和生存力。这些神经元然后FAC直接分类到裂解缓冲液和RNA提取。此协议的意义是定位，隔离和保持一个特定的感觉细胞群体的质量的能力。基因表达在神经元中的这个小群体描述，以及特定器官的功能和神经网络识别。
在这个协议中的关键步骤?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
支持美国国立卫生研究院授予R01HL105635到SEJ。笔者想感谢迭戈五Bohórquez的技术咨询。我们也感谢R.伊恩·卡明技术援助，并在杜克大学人类疫苗研究所流式细胞仪共享资源基金（北卡罗来纳州达勒姆）进行流式细胞术流。流式细胞仪在杜克区域生物控制实验室，收到来自美国国立卫生研究院国家过敏和传染病研究所（UC6-AI058607）建设部分支持执行。
Materials
| Fast Blue | Polysciences, Inc. | 17740-2 | stock 2 mg/ml in water |
| NeuroTrace 530/615 red Nissle stain | Life Technologies | N21482 | |
| Dimethyl Sulfoxide (DMSO) | Fisher Scientific | D128-500 | |
| Dulbecco's Phosphate Buffered Saline (PBS) Ca and Mg free | Gibco | 14190-144 | |
| Advanced DMEM/F12 | Gibco | 12634-010 | |
| glutamine (Glutamax) | Gibco | 35050-061 | |
| HEPES | Gibco | 15630-080 | |
| N2 | Gibco | 17502-048 | |
| B27 (no vitamin A) | Gibco | 12587-010 | |
| Nerve Growth Factor (NGF) | Sigma | N6009 | stock 50 µg/ml in PBS/10% FBS |
| digestion enzyme, Liberase DH Research Grade | Roche | 5401054001 | stock 2.5 mg/ml in water |
| particle solution (Percoll) | Sigma | P1644-25ML | |
| Heating block | LabNet | ||
| 70 um cell strainer | Falcon | 352350 | |
| Absolute Ethanol (200 proof) | Fisher Scientific | BP2818-500 | |
| RNase free water | Fisher Scientific | BP2484-100 | |
| RNase decontamination reagent, RNase AWAY | invitrogen | 10328-011 | |
| 2-mercaptoethanol | VWR | EM-6010 | |
| RNA extraction kit, RNeasy Plus Micro Kit | Qiagen | 74034 | |
| DNase kit, RNase-Free DNase Set | Qiagen | 79254 | |
| DNase | Sigma | D5025-15KU | stock 10 mg/ml in 0.15 M NaCl |
| Propidium Iodide | Sigma | P4170-10MG | stock 10 µg/ml in PBS |
| Microfluidic electrophoresis system (TapeStation 2200) | Agilent |
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