小鼠脊髓小胶质细胞的快速高效富集
Summary
小胶质细胞被认为是体内最通用的细胞之一，能够进行形态和功能适应。它们的异质性和多功能性能够维持大脑稳态，同时也与各种神经系统病理有关。在这里，描述了一种净化脊髓小胶质细胞的技术。
Abstract
脊柱定义了脊椎动物并塑造了椎管，椎管是一个包围和保护脊髓的腔体。哺乳动物中枢神经系统的正常发育和功能在很大程度上依赖于常驻巨噬细胞（称为小胶质细胞）的活性。小胶质细胞具有异质性和多功能性，可在脊髓和大脑内实现不同的基因表达和行为。许多研究探索了脑小胶质细胞的功能，并广泛地详细介绍了纯化方法。然而，小鼠脊髓中小胶质细胞的纯化缺乏全面的描述。相比之下，与未精制的提取物相比，使用高度纯化的胶原酶在中枢神经系统组织中缺乏报告。在这项研究中，从 8-10 周龄的 C57BL/6 小鼠身上切除脊柱和脊髓。随后的消化采用高度纯化的胶原酶，小胶质细胞纯化采用密度梯度。对细胞进行流式细胞术染色，通过 CD11b 和 CD45 染色评估活力和纯度。结果显示，平均存活率为80%，平均纯度为95%。总之，小鼠小胶质细胞的操作涉及用高度纯化的胶原酶消化，然后是密度梯度。这种方法有效地产生了大量的脊髓小胶质细胞群。
Introduction
脊椎动物的定义特征是脊柱或脊柱，其中脊索已被一系列称为椎骨的分段骨骼所取代，这些骨骼被椎间盘分割。这种骨质物质的连续性塑造了椎管，椎管是一个包围和保护脊髓的腔1。在啮齿动物属中，脊柱通常由7个颈椎、13个胸椎、6个腰椎和数量不等的尾椎2,3组成。脊髓的长度与脊柱的长度相似，末端丝是将脊髓固定在骶骨上的非神经结构。此外，神经纤维通过椎间孔1 排出。
哺乳动物中枢神经系统的发育和正常功能在很大程度上取决于神经系统常驻巨噬细胞（称为小胶质细胞4）的活动。尽管小胶质细胞最初被描述为大脑驻留吞噬细胞，但最近的研究将许多动态功能归因于这些细胞 5,6。小胶质细胞的大小在稳态中为 7 至 10 μm;它们被认为是体内用途最广泛的细胞之一，可以在形态和功能上适应不断变化的环境7.这些细胞在胚胎期和成体阶段都表现出高度异质性8,9，而在成体阶段，它们也表现出基于其时空背景的复杂功能异质性10。小胶质细胞的异质性和多种功能允许在脊髓和大脑中产生不同的基因表达和行为。研究表明，与大脑相比，CD11b、CD45、CD86 和 CCR9 在脊髓中的表达更高 8,9。
脑小胶质细胞分离存在多种方案11,12;然而，脊髓小胶质细胞只有少数存在13,14。优化从脊髓中纯化小胶质细胞的方法有助于开展多项专注于发现小胶质细胞生理学的研究。该协议旨在描述小鼠脊髓的简单且高度可重复的提取和小胶质细胞的纯化（图1）。
Protocol
Representative Results
Discussion
由于小胶质细胞在大脑稳态中的重要性，已经开发了许多用于研究小胶质细胞的方案。在这些方法中，小胶质细胞通常来源于胚胎或新生大鼠和小鼠的大脑半球17。有限数量的研究涉及从成年小鼠脊髓中纯化小胶质细胞13,14。这些技术包括使用胶原酶和/或木瓜蛋白酶以及DNAse进行酶消化，通常与使用Percoll或OptiPrep的梯度分离相结合。然?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学技术委员会（CONACYT）（702361）授予的奖学金的支持。作者承认博士国立理工学院国立生物科学学院生物化学科学课程。
Materials
| 15 mL collection tubes | Corning, USA | 430790 | |
| 2 mL microtubes | Axygen, USA | MCT-200-G | |
| 2.4G2 anti-FcR | BioLegend, USA | 101302 | |
| 50 mL collection tubes | Corning, USA | 430829 | |
| 70% ethanol | |||
| Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin b) | Gibco, USA | 15240062 | |
| Antibody CD11b eFluor 450 anti-mouse | eBioscience, USA | 48-0112 | |
| Antibody CD45 PerCP anti-mouse | BioLegend, USA | 103130 | |
| Balanced salt solution (PBS) calcium- magnesium-free | Corning, USA | 46-013-CM | |
| Blue Cell Strainer 40 μm | Corning, USA | 352340 | |
| Costar 6-well Clear Not Treated | Corning, USA | CLS3736 | |
| Coverslips | |||
| Digital Heating Shaking Drybath | Thermo Scientific Digital HS Drybath, USA | 88870001 | |
| Dissecting forceps for microsurgery | FT by DUMONT | ||
| DNase | Roche, USA | 4536282001 | |
| Dulbecco´s Modified Eagle´s Medium-high glucose (DMEM) | Merck, USA | D6429 | |
| Electric shaver | |||
| FACS tube | Thermo, USA | 352058 | |
| Fetal bovine serum (FBS) | PAN Biotech, Alemania | P30-3306 | |
| Flow cytometer Cytoflex | Beckman Coulter | ||
| Hank’s balanced salt solution | Merck, USA | H2387 | |
| L-glutamine | Corning, USA | 15393631 | |
| Liberase TM | Roche, USA | 5401119001 | |
| Neubauer chamber Counting Chambers | China | 1103 | |
| Pentobarbital | |||
| Percoll | Merck, USA | 17089101 | density gradient centrifugation |
| Poly-L-lysine solution | Merck, USA | P8920 | |
| Scalpel No. 25 | HERGOM, Mexico | H23 | |
| Snaplock Microcentrifuge Tubes 2 mL | Axygen, USA | 10011-680 | |
| Stereoscopic microscope | Velab, Mexico | HG927831 | |
| Straight surgical scissors (10 cm) | HERGOM, Mexico | ||
| Straight Vannas scissors | HERGOM, Mexico | ||
| Triton X100 | Merck, USA | X100 | |
| Trypan blue Stain 0.4% | Merck, USA | 15250-061 | |
| Vortex mixer | DLAB, China | 8031102000 | |
| Zombie Aqua Fixable Viability Kit | BioLegend, USA | 423102 | amine-reactive fluorescent dye staining |
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