脊髓神经元从新生小鼠中分离和培养
Summary
这项研究提出了一种从WT新生小鼠中分离神经元的技术。它需要从新生小鼠中仔细地解剖脊髓，然后通过机械和酶切割将脊髓组织中的神经元分离出来。
Abstract
我们提出了脊髓神经元分离和培养的方案。神经元从新生儿C57BL / 6小鼠获得，并在出生后第1-3天分离。一只小鼠垫，通常从一个繁殖对出生的4-10只小狗被聚集一个实验，并且在用异氟烷治疗安乐死后，从每只小鼠单独收集脊髓。将脊柱解剖出来，然后将脊髓从柱上释放出来。然后将脊髓切碎以增加允许神经元和其他细胞从组织中释放的酶蛋白酶的递送的表面积。然后使用研磨将细胞释放到溶液中。随后将该溶液以密度梯度分级，以分离溶液中的各种细胞，允许分离神经元。从一个垃圾组可以分离出约1-2.5×10 6个神经元。然后将神经元接种到涂有粘合剂的孔上rs允许适当的成长和成熟。神经元在生长和培养基中需要约7天达到成熟度，此后可用于治疗和分析。
Introduction
了解脊髓病理学需要在宏观和微观层面上使用各种模型。大型和小型动物模型1,2,3用于脊髓疾病和损伤的体内研究。 在体内研究这些问题的优点是脊髓分析局限于全脊髓匀浆或组织切片4 。当在其驻留神经元和周围神经胶质中分离脊髓中的特定反应和目标时，这产生了一些歧义。遗传操纵的小鼠越来越多的可用性允许对细胞和分子水平的生物学进行更详细的研究。因此，这里使用新生儿小鼠模型，允许在体外研究脊髓神经元的独特性质和生物学。
在体外分离和维持神经元并不是特别简单，成年啮齿动物的皮层组织的神经元分离技术相对丰富，似乎导致大量的孤立神经元（ 即数百万） 5 ，相反，由于组织质量较小，脊髓组织中神经元的产量较低8,9,10 ，另外在小鼠中，新生儿隔离技术相对较少脊髓神经元和现有的方法受到较低的神经元产量（ 即数百个）的限制9或需要分离胚胎小鼠的费力和资源性重的技术10 。在这个协议中，我们使用一种技术，允许从新生小鼠脊髓的成本和资源有效的隔离大量的神经元。如以前发表的技术中常见的，我们使用木瓜蛋白酶作为酶蛋白酶，允许从脊髓组织释放神经元5,6 。此外，我们使用密度梯度进行精细细胞分离，其先前已被证明是有效的6,10 。虽然培养细胞的培养基可以根据我们的经验和以前发表的11来改变 ，但补充新鲜的B27培养基补充剂已被证明对神经元寿命至关重要。神经元通常可行达10天，允许进行治疗。
Protocol
根据科罗拉多大学机构动物护理和使用委员会的指导方针，对该手术中的动物进行护理和治疗。 准备解决方案在适当的温度下准备并储存所有溶液，如表1所示。 2.涂层孔和滑块注意：神经元不能很好地粘附在塑料或玻璃表面上。 在分离神经元前一天，用无菌的24孔培养板的孔用0.5mL聚-D-赖氨酸（PDL; <s…
Representative Results
使用这种技术，单个垃圾（4-10只幼仔）允许分离适合种植在培养板上的1-2.5个10 6个神经元。通常，以上述浓度（ 即 30万个细胞/ mL）接种4-8个孔。 图3显示了在低（ a ）和高（ b ）放大光学显微镜下培养一周后在该浓度下神经元的出现。然而，我们也能够以高达500,000个细胞/孔和低至100,000个细胞/孔的浓度培养?…
Discussion
这种技术允许脊髓神经元的可靠培养。一旦熟练掌握了这项技术，大概需要3.5h才能完成。我们已经能够在大约4小时内从2个独立的小窝（总共16只小鼠）中进行神经元的分离。可行性的关键步骤是能够精通从小鼠中提取脊髓。产量允许电镀几个孔，并能够在各种条件下测试神经元。我们已经能够在成熟后治疗神经元，并使用蛋白质印迹分析可靠地评估蛋白质表达。此外，在孔内使用玻璃载玻片进?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者没有确认。
Materials
| Hibernate A Medium – 500 mL | Thermo-Fisher | A1247501 | https://www.thermofisher.com/order/catalog/product/A1247501 |
| Hibernate A Minus Calcium – 500 mL | Brainbits | HA-Ca | http://www.brainbitsllc.com/hibernate-a-minus-calcium/ |
| Glutamax 100X – 100 mL | Thermo-Fisher | 35050061 | https://www.thermofisher.com/order/catalog/product/35050079 |
| B27 Supplement 50X – 10 mL | Thermo-Fisher | 17504044 | https://www.thermofisher.com/order/catalog/product/17504044 |
| Papain, Lyophilized – 100 mg | Worthington | LS003119 | http://www.worthington-biochem.com/pap/cat.html |
| Neurobasal A Medium – 500 mL | Thermo-Fisher | 10888022 | https://www.thermofisher.com/order/catalog/product/10888022 |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo-Fisher | 15140122 | https://www.thermofisher.com/order/catalog/product/15140122 |
| Poly-D-lysine hydrobromide – 5 mg | Sigma-Aldrich | P6407-5MG | http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en®ion=US |
| Mouse Laminin – 1 mg | Thermo-Fisher | 23017015 | https://www.thermofisher.com/order/catalog/product/23017015 |
| Trypan Blue – 20 mL | Sigma-Aldrich | T8154-20ML | http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en®ion=US |
| OptiPrep Density Gradient Medium – 250 mL | Sigma-Aldrich | D1556-250ML | http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en®ion=US |
| Dichlorodimethylsilane (DMDCS, Sigma Silicoat) | Sigma-Aldrich | 440272-100ML | http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en®ion=US |
| Chloroform | Sigma-Aldrich | 288306-1L | http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en®ion=US |
| Glass Pippette – 9" | Sigma-Aldrich | 13-678-20C | http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en®ion=US |
| Pipette bulb – 5 mL | Sigma-Aldrich | Z186678-3EA | http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1 |
| BRAND® Petri dish, glass – 60×15 mm | Sigma-Aldrich | BR455717-10EA | http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en®ion=US |
| Sterile 24 Well Cell Culture Plate | Sigma-Aldrich | M8812-100EA | http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en®ion=US |
| Hausser Hemacytometer (glass counting chamber) | Fischer Scientific | 02-671-6 | https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716 |
| Glass Slides – 12 mm sterile cover glass – uncoated | Neuvitro | GG-12-1.5-Pre | http://www.neuvitro.com/german-coverslip-12mm-diameter.htm |
| NeuN Rabbit Monoclonal Antibody – 100 µL | Abcam | ab177487 | After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:200 for 18 hours in 4 °C |
| MAP-2 Mouse Monoclonal Antibody – 50 µL | Abcam | ab11267 | After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:500 for 18 hours in 4 °C |
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Citazione di questo articolo
Eldeiry, M., Yamanaka, K., Reece, T. B., Aftab, M. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (125), e55856, doi:10.3791/55856 (2017).