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Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue
从CNS组织分离单核细胞及FACS分析   

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Abstract

Immune cells, such as microglia are resident in the brain and spinal cord of normal mice and humans. Furthermore, macrophages, dendritic cells, T cells, B cells and NK cells infiltrate the CNS during certain infections or in neurodegenerative/neuroinflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) (a model for multiple sclerosis) or Alzheimer’s disease (Sutton et al., 2009; Browne et al., 2013). Infiltrating cells can be identified using immunohistological staining of sections from brain or spinal cords. However, more detailed phenotypic and functional analysis is possible following isolation of the immune cells from the CNS of normal or diseased mice. Purification of mononuclear cells from brain or spinal cord is dependent on perfusing the mouse to ensure removal of the blood from the CNS tissue, prior to dissociating the tissue and purification of the mononuclear cells on a percoll gradient. The technique provides single cell suspensions with cells of high viability that are suitable for FACS analysis or limited functional studies. The yields are usually low from the normal mouse brain or spinal cord, but higher from mice with EAE or CNS infection. When combined with intracellular cytokine staining and FACS, this technique is particularly useful for analysis of the pathogenic T cells (Th17 and Th1 cells) and their regulation/modulation in EAE.

Materials and Reagents

  1. Mice (adult >6 weeks, any strain, e.g. C57BL/6 used for MOG-induced EAE)
  2. Sodium pentobarbital (euthatal) (Merial)
  3. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
  4. 10x PBS (Sigma-Aldrich, catalog number: D1408 )
  5. RPMI solution(Sigma-Aldrich, catalog number: R0883 )
  6. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333 )
  7. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  8. FBS (Sigma-Aldrich, catalog number: F9665 )
  9. Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9394 ) supplemented with 3% FBS
  10. Collagenase D (Roche, catalog number: 11088858001 )
  11. DNase I (Sigma-Aldrich, catalog number: D4263 )
  12. Percoll Plus (Sigma-Aldrich, catalog number: 17-5445-01 )
  13. Cell permeabilisation kit (contains IntraStain Reagent A and B) (Dako, Denmark, catalog number: K2311 )
  14. Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P1585 )
  15. Ionomycin (Sigma-Aldrich, catalog number: I0634 )
  16. Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651 )
  17. LIVE/DEAD® Fixable Aqua Dead Cell Stain kit (Life Technologies, catalog number: L34957 )
  18. CD16/CD32 FcγRIII (BD Biosciences, catalog number: 553141 )
  19. FACS antibodies (as appropriate)
  20. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864 )
  21. Complete RPMI solution (see Recipes)
  22. FACS buffer (see Recipes)
  23. Stock isotonic percoll (SIP) (see Recipes).

Equipment

  1. 70 μm nylon mesh filter (Corning, catalog number: 352350 )
  2. Shaker capable of 200 rpm at 37 °C
  3. Tissue culture facilities including class II laminar flow hood
  4. Bench-top centrifuge , preferably refrigerated (any model)
  5. Flow cytometer

Software

  1. Summit software (Dako)
  2. FlowJo software (Tree Star)

Procedure

  1. Anaesthetise mice with sodium pentobarbital (40 μl i.p. ) and perfuse intracardially with sterile ice-cold PBS (20 ml). This is achieved by slowly and steadily injecting the ice-cold PBS into the left ventricle of the heart using a 20 ml syringe.
  2. Dissect out brains by cutting away the skull with a sharp scissors, gently removing the brain with a forceps and place in 1 ml complete RPMI solution (or HBSS supplemented with 3% FBS can be used).
  3. Prepare a single cell suspension by forcing the tissue through a sterile 70 μm nylon mesh filter using the plunger of a 5 ml syringe, wash with complete RPMI solution and centrifuge at 280 x g for 5 min.
  4. Remove the supernatant and the remaining pellet and resuspend in complete RPMI (2 ml) containing collagenase D (1 mg/ml) and DNAse I (10 μg/ml), and incubate for 1 h at 37 °C with agitation.
  5. Wash cells with complete RPMI and centrifuge at 280 x g for 5 min. Discard supernatants and resuspend cells in 40% Percoll [5 ml; 40% stock isotonic percoll (SIP) in PBS; 1.052 g/ml].
  6. Carefully layer cell suspension on top of 70% Percoll (5 ml; 70% SIP in PBS; 1.088 g/ml).
  7. Centrifuged at 600 x g for 20 min with the brake of the centrifuge switched off.
  8. Remove mononuclear cells from the 1.088:1.052 g/ml interface by aspiration with sterile plastic pipette, wash twice in complete RPMI and count.
  9. Samples are centrifuged at 280 x g for 5 min and cells are incubated in sterile FACS tubes at 37 °C in complete RPMI in the presence of PMA (10 ng/ml), ionomycin (1 μg/ml) and BFA (5 μg/ml) for 5 h.
  10. After 5 h wash the cells by centrifuging the cells at 280 x g for 5 min, ready for intracellular staining using a cell permeabilisation kit.
  11. Resuspend in 50 μl PBS with 1:1,000 LIVE/DEAD® Fixable Aqua Dead Cell Stain kit for 20 min at 4 °C.
  12. Wash cells in FACS buffer and resuspend in 50 μl FACS buffer containing CD16/CD32 FcγRIII (1: 100) and incubate at 4 °C for 10 min to block low-affinity IgG receptors.
  13. Incubate cells in 50 μl/sample FACS buffer containing the appropriate FACS antibodies for 15 min at 4 °C.
  14. Fix cells using IntraStain Reagent A (50 μl/sample) for 15 min at RT, wash twice with FACS buffer and centrifuge at 280 x g for 5 min.
  15. Permeabilise cells with IntraStain Reagent B (50 μl/sample) including intracellular antibodies for 15 min at room temperature in the dark.
  16. Wash cells twice in FACS buffer and centrifuge at 280 x g for 5 min.
  17. Mononuclear cells which were surface stained only are blocked for 10 min, incubated with the appropriate antibodies for 15 min at 4 °C as described above, washed twice in FACS buffer and centrifuged at 280 x g for 5 min. PI can be used as a live/dead stain (with surface staining only) by adding 1:100 immediately before reading the samples on a flow cytometer.
  18. Perform flow cytometric analysis on a flow cytometer and acquire data using Summit software. Analyse results using FlowJo software. Representative data can be viewed in References 1 and 2 below.

Recipes

  1. Complete RPMI solution
    RPMI solution supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% FBS
    Note: HBSS supplemented with 3% FBS can be used as a substitute for complete RPMI solution.
  2. FACS buffer
    PBS supplemented with 2% FBS
  3. Stock isotonic Percoll (SIP)
    9 parts Percoll plus (from the bottle), 1 part 10x PBS

Acknowledgments

This work was funded by a PI grant to Kingston Mills from Science Foundation Ireland.

References

  1. Browne, T. C., McQuillan, K., McManus, R. M., O'Reilly, J. A., Mills, K. H. and Lynch, M. A. (2013). IFN-gamma production by amyloid beta-specific Th1 cells promotes microglial activation and increases plaque burden in a mouse model of Alzheimer's disease. J Immunol 190(5): 2241-2251.
  2. Sutton, C. E., Lalor, S. J., Sweeney, C. M., Brereton, C. F., Lavelle, E. C. and Mills, K. H. (2009). Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 31(2): 331-341.

简介

免疫细胞,例如小神经胶质细胞存在于正常小鼠和人的脑和脊髓中。此外,巨噬细胞,树突细胞,T细胞,B细胞和NK细胞在某些感染或神经变性/神经炎症疾病如实验性自身免疫性脑脊髓炎(EAE)(多发性硬化症模型)或阿尔茨海默病(Sutton >等人,2009; Browne等人,2013)。可以使用来自脑或脊髓的切片的免疫组织学染色来鉴定浸润细胞。然而,在从正常或患病小鼠的CNS中分离免疫细胞后,可能进行更详细的表型和功能分析。来自脑或脊髓的单核细胞的纯化取决于灌注小鼠以确保在离解组织和在Percoll梯度上纯化单核细胞之前从CNS组织中除去血液。该技术提供了具有高存活力的细胞的单细胞悬浮液,其适合于FACS分析或有限的功能研究。产率通常低于正常小鼠脑或脊髓,但从具有EAE或CNS感染的小鼠的产量更高。当与细胞内细胞因子染色和FACS组合时,该技术特别用于分析致病性T细胞(Th17和Th1细胞)及其在EAE中的调节/调节。

材料和试剂

  1. 小鼠(成年人> 6周,任何菌株,例如用于MOG诱导的EAE的C57BL/6)
  2. 戊巴比妥钠(euthatal)(Merial)
  3. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537)
  4. 10x PBS(Sigma-Aldrich,目录号:D1408)
  5. RPMI溶液(Sigma-Aldrich,目录号:R0883)
  6. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P4333)
  7. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  8. FBS(Sigma-Aldrich,目录号:F9665)
  9. 补充有3%FBS的Hank's平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H9394)
  10. 胶原酶D(Roche,目录号:11088858001)
  11. DNase I(Sigma-Aldrich,目录号:D4263)
  12. Percoll Plus(Sigma-Aldrich,目录号:17-5445-01)
  13. 细胞渗透试剂盒(含有IntraStain试剂A和B)(Dako,丹麦,目录号:K2311)
  14. 佛波醇肉豆蔻酸乙酯(PMA)(Sigma-Aldrich,目录号:P1585)
  15. 离子霉素(Sigma-Aldrich,目录号:I0634)
  16. 布雷菲德菌素A(BFA)(Sigma-Aldrich,目录号:B7651)
  17. LIVE/DEAD ® Fixable Aqua死细胞染色试剂盒(Life Technologies,目录号:L34957)
  18. CD16/CD32FcγRIII(BD Biosciences,目录号:553141)
  19. FACS抗体(视情况而定)
  20. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4864)
  21. 完成RPMI解决方案(参见配方)
  22. FACS缓冲区(参见配方)
  23. 原料等渗percoll(SIP)(参见Recipes)。

设备

  1. 70μm尼龙网过滤器(Corning,目录号:352350)
  2. 振荡器,在37℃下能够达到200rpm。
  3. 组织培养设施包括II级层流罩
  4. 台式离心机,优选冷藏(任何型号)
  5. 流式细胞仪

软件

  1. Summit软件(Dako)
  2. FlowJo软件(Tree Star)

程序

  1. 用戊巴比妥钠麻醉麻醉小鼠(40μli.p.)并用无菌冰冷PBS(20ml)心内灌注。 这是通过使用20ml注射器缓慢且稳定地将冰冷的PBS注射到心脏的左心室中来实现的。
  2. 通过用锋利的剪刀切除头骨,用镊子轻轻地除去大脑,并放置在1ml​​完全RPMI溶液(或可以使用补充3%FBS的HBSS)中解剖大脑。
  3. 通过使用5ml注射器的柱塞迫使组织通过无菌的70μm尼龙网过滤器,用完全RPMI溶液洗涤并以280×g离心5分钟来制备单细胞悬浮液。
  4. 除去上清液和剩余的沉淀并重悬于含有胶原酶D(1mg/ml)和DNAse I(10μg/ml)的完全RPMI(2ml)中,并在37℃下搅拌温育1小时。
  5. 用完全RPMI洗涤细胞并在280×g离心5分钟。弃去上清液并将细胞重悬于40%Percoll [5ml; PBS中的40%储备等渗percoll(SIP); 1.052g/ml]。
  6. 小心地在70%Percoll(5ml; 70%SIP在PBS中; 1.088g/ml)顶上的细胞悬浮液。
  7. 离心机关闭制动器时,以600 x g离心20分钟。
  8. 通过用无菌塑料移液管吸出,从1.088:1.052 g/ml接口中取出单核细胞,在完全RPMI中洗涤两次并计数。
  9. 将样品以280×g离心5分钟,并在存在PMA(10ng/ml),离子霉素(1μg/ml)的情况下,将细胞在37℃下在无菌FACS管中在完全RPMI中温育。和BFA(5μg/ml)处理5小时
  10. 5小时后,通过在280×g离心细胞5分钟来洗涤细胞,准备使用细胞透化试剂盒进行细胞内染色。
  11. 重悬在50μlPBS与1:1,000 LIVE/DEAD可固定水族死细胞染色试剂盒在4℃下20分钟。
  12. 在FACS缓冲液中洗涤细胞,并重悬于含有CD16/CD32FcγRIII(1:100)的50μlFACS缓冲液中,并在4℃下孵育10分钟以封闭低亲和力IgG受体。
  13. 孵育细胞在50微升/含有适当的FACS抗体的样品FACS缓冲液在4℃下15分钟
  14. 使用IntraStain试剂A(50μl/样品)在室温下固定细胞15分钟,用FACS缓冲液洗涤两次并在280×g离心5分钟。
  15. 使用IntraStain试剂B(50μl/样品)包括细胞内抗体在室温下在黑暗中透过细胞15分钟。
  16. 在FACS缓冲液中洗涤细胞两次,并在280×g离心5分钟。
  17. 仅将表面染色的单核细胞阻断10分钟,如上所述在4℃下用合适的抗体温育15分钟,在FACS缓冲液中洗涤两次,并在280×g离心5分钟。 PI可以在流式细胞仪上读取样品之前立即通过加入1:100来用作活/死染色(仅具有表面染色)。
  18. 在流式细胞仪上进行流式细胞术分析,并使用Summit软件获取数据。 使用FlowJo软件分析结果。 代表性数据可参见下面的参考文献1和2。

食谱

  1. 完成RPMI解决方案
    补充有1%青霉素 - 链霉素,1%L-谷氨酰胺和10%FBS的RPMI溶液 注意:补充了3%FBS的HBSS可以代替完整的RPMI溶液。
  2. FACS缓冲区
    补充有2%FBS的PBS
  3. 股用等渗Percoll(SIP)
    9份Percoll加(来自瓶),1份10x PBS

致谢

这项工作是由科学基金会爱尔兰的金斯顿米尔斯的PI拨款资助。

参考文献

  1. Browne,T.C.,McQuillan,K.,McManus,R.M.,O'Reilly,J.A.,Mills,K.H.and Lynch,M.A。(2013)。 淀粉状蛋白β特异性Th1细胞的IFN-γ产生促进小胶质细胞活化并增加小鼠的斑块负荷 模型的阿尔茨海默病。 Immunol 190(5):2241-2251。
  2. Sutton,C.E.,Lalor,S.J.,Sweeney,C.M.,Brereton,C.F.,Lavelle,E.C。和Mills,K.H。(2009)。 白细胞介素-1和IL-23诱导来自gammadelta T细胞的天然IL-17产生,扩增Th17应答 和自身免疫。 免疫 31(2):331-341。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mills, K. H., McManus, R. M. and Dungan, L. (2014). Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue. Bio-protocol 4(18): e1240. DOI: 10.21769/BioProtoc.1240.
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