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Isolation of Infiltrating Leukocytes from the Spinal Cord of Mice
从小鼠脊髓分离浸润性白细胞

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Abstract

The infiltration of leukocytes into the central nervous system (CNS) is a common feature of many neuroinflammatory diseases such as multiple sclerosis, and also occurs during certain microbial infections such as by West Nile Virus. Here, we describe a method to isolate leukocytes from the spinal cords of mice. This method can be used for the characterization of leukocyte populations that infiltrate the spinal cord, and to perform functional studies with the isolated cells. The CNS of naive mice is infiltrated by very low numbers of leukocytes, however, upon inflammation increased numbers of mononuclear cells traffic to the CNS. The number of leukocytes that can be isolated roughly correlates with the degree of inflammation.

Keywords: Experimental Autoimmune Encepahlomyelitis(实验性自身免疫性脑脊髓炎), CD137(CD137), 4-1BB(4-1BB), Infiltration(浸润), Spinal cord(脊髓)

Materials and Reagents

  1. 1x PBS and 10x PBS
  2. Surgical instruments: scissors, forceps, scalpel
  3. Percoll (Sigma-Aldrich, catalog number: P4937 )
    Note: prepare isotonic Percoll solution stock (one part 10x PBS and 9 parts Percoll).

Equipment

  1. Centrifuge with swing-out bucket rotor (e.g. Eppendorf, model: 5810 R )
  2. 75 mm petri dish
  3. 18 G and 27 G needle (BD Biosciences)
  4. 10 ml syringe (BD Biosciences)
  5. 15 and 50 ml conical centrifuge tubes
  6. Plastic Pasteur pipette
  7. 70 μm cell strainer (BD Biosciences, catalog number: 352350

Procedure

  1. Preparation of the animal
    1. Euthanize the mouse using CO2 gas (cervical dislocation might damage the spinal cord therefore it should be avoided).
    2. Spray the mouse with 70% ethanol to maintain aseptic conditions and cut open the chest wall to expose the heart.

  2. Perfusion of the animal
    1. Attach a 27G needle to a 10 ml syringe pre-filled with ice-cold PBS.
    2. Cut the right atrium of the heart with scissors (Figure 1). 
    3. Perfuse the animal with the entire 10 ml of PBS through the left ventricle (Figure 1), repeat this procedure for 3-5 times (final volume of ~50 ml). Alternatively, a perfusion pump can be used in this step. By the end of the perfusion, the effluent from the atrium should be clear and the liver should have lost its red color. If this does not occur, continue to perfuse.


      Figure 1. Photograph of the heart with the incision site (right atrium) and injection site (left ventricle) indicted.

  3. Isolation of the spinal cord
    1. Spray the back of the mouse with ethanol. Remove the head using scissors by doing a clean cut at the base of the head (start of the spinal column). Then expose the spinal column by cutting open the skin on the back from the base of the head to the tail of the animal. Identify where the base of the spinal column attaches to the pelvis.
    2. Using a pair of scissors, make a perpendicular cut through the spinal column at this location.
    3. Attach an 18G needle to a 10 ml syringe pre-filled with PBS. Hold the spinal column with forceps and identify the spinal canal at the caudal end of the spinal column. Carefully insert the 18G needle up to 10 mm at this point. Resistance will be encountered as the needle enters the spinal canal.
    4. Slowly push past the resistance in the canal until the point where the resistance is suddenly lost. Hold the spinal column over the 75 mm petri dish and flush the spinal cord out of the column.

  4. Dissociation of the spinal cord tissue
    1. Transfer the spinal cords to a 50 ml conical centrifuge tube containing 1x PBS and keep on ice (pool a maximum of 5 spinal cords per tube). In a petri dish and using scalpel or scissors and a pair of forceps, cut the spinal cords into very small pieces of about 10 mm.
    2. Transfer the spinal cord pieces into a 70 µm cell strainer placed on top of a 50 ml conical centrifuge tube and homogenize the tissue using the plunger of a 5 ml syringe. Add cold PBS to wash the cell strainer and top up to 50 ml.
    3. Centrifuge at 400 x g for 10 min, discard the supernatant and resuspend the cells in 5 ml of 30% isotonic Percoll (prepared using Percoll stock solution and 1x PBS). Add the 30% isotonic Percoll gently on top of 4 ml 70% isotonic Percoll (prepared using Percoll stock solution and 1x PBS) contained in a 15 ml conical centrifuge tube. Avoid mixing of the interface between the 70% and 30% solutions. Percoll should be used at room temperature, since cold cells tend to clump.
    4. Centrifuge for 20 min at 500 x g, at room temperature, without break or accelerator. The myelin will form a compact layer at the top, while the leukocytes will form an opaque ring between the two layers of Percoll, the so called interface.
    5. Remove the myelin and part of the supernatant above the interface by aspiration.
    6. Collect the cells in the interface into a clean 15 ml conical tube. Wash the cells 1-2x with 1x PBS by centrifuging at 400 x g for 5 min. Proceed with any further analysis of experimentation. If culturing of the cells is required, processing of the spinal cords (step IV) should be performed under sterile conditions, e.g. in a biological biosafety cabinet.

Acknowledgments

This protocol is was adapted from and utilized in a publication by Martinez Gomez et al. (2012).

References

  1. Martinez Gomez, J. M., Croxford, J. L., Yeo, K. P., Angeli, V., Schwarz, H. and Gasser, S. (2012). Development of experimental autoimmune encephalomyelitis critically depends on CD137 ligand signaling. J Neurosci 32(50): 18246-18252.

简介

白细胞向中枢神经系统(CNS)的渗透是许多神经炎症性疾病如多发性硬化症的常见特征,并且也发生在某些微生物感染(例如西尼罗病毒)中。 在这里,我们描述了一种从小鼠脊髓分离白细胞的方法。 该方法可用于表征渗透脊髓的白细胞群体,并对分离的细胞进行功能研究。 幼稚小鼠的CNS被非常少量的白细胞渗透,然而,在炎症时,单核细胞数量增加到CNS的流量。 可分离的白细胞数量与炎症程度大致相关。

关键字:实验性自身免疫性脑脊髓炎, CD137, 4-1BB, 浸润, 脊髓

材料和试剂

  1. 1x PBS和10x PBS
  2. 外科器械:剪刀,镊子,手术刀
  3. Percoll(Sigma-Aldrich,目录号:P4937) 注意:制备等渗Percoll溶液原液(一份10x PBS和9份Percoll)。

设备

  1. 使用转出式转子离心机(例如Eppendorf,型号:5810R)
  2. 75 mm培养皿
  3. 18 G和27 G针(BD Biosciences)
  4. 10ml注射器(BD Biosciences)
  5. 15和50ml锥形离心管
  6. 塑料巴斯德吸管
  7. 70μm细胞过滤器(BD Biosciences,目录号:352350)<

程序

  1. 动物的制备
    1. 使用CO 2气体安乐死小鼠(颈部错位可能损害脊髓,因此应该避免)。
    2. 用70%乙醇喷洒小鼠以保持无菌条件,并切开胸壁以暴露心脏
  2. 动物灌注
    1. 将27G针连接到预先填充有冰冷PBS的10ml注射器中。
    2. 用剪刀剪下心脏的右心房(图1)。
    3. 灌注动物与整个10毫升PBS穿过左心室(图1),重复此过程3-5次(最终体积〜50毫升)。 或者,在该步骤中可以使用灌注泵。 在灌注结束时,来自心房的流出物应该清澈,肝脏应该失去其红色。 如果这不发生,继续灌注。


      图1.带有切口部位(右心房)和注射部位(左心室)的心脏照片 。
  3. 脊髓的隔离
    1. 用乙醇喷洒小鼠的背部。使用剪刀通过在头部的基部(脊柱开始的一个干净的切口)去除头。然后通过切开背部皮肤从头部的基部到动物的尾部暴露脊柱。识别脊柱的基部附着到骨盆的位置。
    2. 使用一把剪刀,在这个位置做一个垂直切割通过脊柱。
    3. 将18G针附接到预填充有PBS的10ml注射器。用镊子握住脊柱,并确定脊柱尾部的脊柱。在这一点上小心地将18G针插入到10mm。当针进入椎管时将遇到阻力。
    4. 慢慢地推过运河中的阻力,直到阻力突然消失的地方。将脊柱保持在75 mm培养皿上,将脊髓从柱中冲洗出来。

  4. 脊髓组织的解离
    1. 将脊髓转移到含有1×PBS的50 ml锥形离心管,并保持在冰上(每管最多5根脊髓)。在培养皿中,使用手术刀或剪刀和一对镊子,将脊髓切成约10毫米的非常小的片。
    2. 将脊髓片转移到放置在50ml锥形离心管顶部的70微米细胞过滤器,并使用5ml注射器的柱塞匀浆组织。加入冷PBS洗涤细胞过滤器和补充至50毫升。
    3. 在400×g离心10分钟,弃去上清液并将细胞重悬于5ml的30%等渗Percoll(使用Percoll储备溶液和1×PBS制备)中。在15ml锥形离心管中包含的4ml 70%等渗Percoll(使用Percoll储备溶液和1×PBS制备)的顶部上轻轻添加30%等渗Percoll。避免混合70%和30%溶液之间的界面。 Percoll应在室温下使用,因为冷的细胞趋于聚集。
    4. 在室温下在500×g离心20分钟,没有断裂或加速器。髓鞘将在顶部形成致密层,而白细胞将在两者之间形成不透明环 层的Percoll,所谓的界面。
    5. 通过抽吸除去髓鞘和一部分的界面上方的上清液。
    6. 收集细胞在界面中的一个干净的15毫升锥形管。 通过在400×g离心5分钟用1x PBS洗涤细胞1-2x。 继续进行任何进一步的实验分析。 如果需要培养细胞,则应在无菌条件下,例如在生物生物安全柜中进行脊髓的加工(步骤IV)。

致谢

该方案改编自Martinez Gomez等人的出版物并在其中使用。 (2012)。

参考文献

  1. Martinez Gomez,J.M.,Croxford,J.L.,Yeo,K.P.,Angeli,V.,Schwarz,H。和Gasser,S。(2012)。 实验性自身免疫性脑脊髓炎的发展关键取决于CD137配体信号传导。 J Neurosci 32(50):18246-18252。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Martinez Gomez, J. M., Gasser, S. and Schwarz, H. (2013). Isolation of Infiltrating Leukocytes from the Spinal Cord of Mice. Bio-protocol 3(10): e775. DOI: 10.21769/BioProtoc.775.
  2. Martinez Gomez, J. M., Croxford, J. L., Yeo, K. P., Angeli, V., Schwarz, H. and Gasser, S. (2012). Development of experimental autoimmune encephalomyelitis critically depends on CD137 ligand signaling. J Neurosci 32(50): 18246-18252.
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