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Isolation of CNS-infiltrating and Resident Microglial Cells
中枢神经系统浸润和固有小胶质细胞的分离   

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Abstract

Variety of immunological and biochemical studies associated with infection or inflammation in the central nervous system (CNS) utilize CNS-resident and/or infiltrating cells which were isolated from the CNS of naïve and affected mice in order to investigate the underlying mechanisms and the potential roles of the cell populations. Mechanical and enzyme-based single cell preparations of CNS cells are subjected to a density gradient to obtain functional single cells. In combination with cell-specific biomarkers, the function and/or status of resident microglia and infiltrating lymphocytes, including B and T cells as well as macrophages, can be characterized.

Materials and Reagents

  1. Isoflurane (Vedco, catalog number: 50201 )
  2. Hanks' balanced salt solution (HBSS) (Cellgro, catalog number: 20-021-CV )
    Note: 10x solution, diluted to 1x in house in sterile distilled water.
  3. RPMI-1640 medium (Sigma-Aldrich, catalog number: R8758 )
  4. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11055H )
  5. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  6. Collagenase type IV (Worthington Biochemical, catalog number: 4188 )
  7. DNase I (Sigma-Aldrich, catalog number: DN25 )
  8. Percoll (GE Healthcare, catalog number: 17-0891-01 )
  9. Phosphate buffered saline (PBS)
  10. Anti-CD45-allophycocyanin (APC) and anti-CD11b-phycoerythrin (PE) antibodies (both from BD, catalog numbers: 559864 and 557397 , respectively)
  11. Anti-CD8-APC (clone Ly-; 2) (BD, catalog number: 553035 )
  12. Anti-CD4-PE (clone L3T4) (BD, catalog number: 553049 ) antibodies
  13. Complete RPMI-1640 medium (see Recipes)
  14. Collagenase and DNase I solution (see Recipes)
  15. 100% Percoll (see Recipes)
  16. 70% Percoll (see Recipes)
  17. 30% Percoll (see Recipes)

Equipment

  1. Scissors and forceps
  2. 10 ml and 50 ml disposable plastic syringe (BD)
  3. 18 G needle (BD)
  4. Steel mesh (250 m) (VWR International, catalog number: AA13478-RR )
  5. 37 °C water bath
  6. Table top refrigerated centrifuge
  7. High-speed centrifuge (Beckman Coulter, model: J2-21 )
  8. 50 ml screw-cap tube
  9. LSRII Flow cytometer (BD)
  10. Low-speed table top centrifuge (1,000-3,000 rpm)

Procedure

  1. Naïve and virus (Theiler’s Murine Encephalomyelitis Virus)-infected mice (3-5 vs. 2-3, respectively) are anesthetized by Isoflurane at 7 days post infection.
  2. Anesthetized mice are transcardially perfused with 30 ml of sterile HBSS through left ventricle using 50 ml syringe.
  3. Decapitate the mice and excise the brain. Flush the spinal cord from the spinal column using the 10 ml syringe filled with HBSS.
  4. Excised brains and spinal cords are grinded separately or together using rubber-attached disposable syringe plunger through a steel mesh to prepare single-cells suspensions.
  5. Grinded brain and spinal cords are subjected to 300 μg/ml collagenase type IV plus 20 μg/ml and incubated at 37 °C for 45 min.
  6. Carefully decant the collagenase-containing supernatant after spinning the cell suspension at 630 x g (1,800 rpm) for 10 min at room temperature.

    Isolation of CNS infiltrating cells using continuous gradient
    A continuous 100% Percoll gradient is used to enrich CNS mononuclear cells. 10 ml of 100% Percoll is added to 20 ml of cell suspension in complete RPMI and then gently mixed by inverting the tube. Centifuge without braking, at 25,000 x g (15,000 rpm) and 37 °C for 30 min using a J2-21 centrifuge. Collect mononuclear cells in the bottom one-third by aspirating out the cell debries in the top 15-20 ml.
  7. Transfer the cells in the bottom Percoll gradient (10-15 ml) to v-bottom 50 ml tube.
  8. Bring up the volume to 50ml with HBSS or RMPI, gently mix and then centrifuge at 630 x g (1,800 rpm) for 10 min.
  9. Resuspend cells in complete RPMI medium.
  10. Approximately 1-2 x 106 cells are obtained from the brain and spinal cord per mouse at 7 days post TMEV infection. Approximately 2-5 x 105 cells are obtained from the CNS of a naïve mouse.

    Isolation of CNS infiltrating cells using discontinuous gradient
    Alternatively, microglia and infiltrating mononuclear cells are isolated at the interface of 70% and 30% Percoll. In particular, microglia from naïve mice are isolated using this more defined method. Single-cell suspensions of 3-5 brains and spinal cords from naïve mice or 2-3 brains and spinal cords from infected mice, which are prepared as described above (steps 1-6), are subjected to a discontinuous Percoll gradient (70 and 30%) at 2,800 x g (5,000 rpm) and 20 °C for 20 min (resuspend cells in 15 ml 30% percoll and underlay 20 ml 70% Percoll). The cells that accumulated in the interface of the gradient are collected and transferred to v-bottom 50 ml tube to wash.
  11. Isolated CNS-infiltrating mononuclear cells (5 x 105) are stained and analyzed by flow cytometry after gating live cells based on FSC and SSC (see Notes). The expression of CD45 and CD11b was measured using anti-CD45-allophycocyanin (APC) and anti-CD11b-phycoerythrin (PE) antibodies to distinguish CNS-resident CD45int microglia from infiltrating CD45hi leukocytes, in conjunction with their expression of CD11b (macrophages) or lack of it (lymphocytes). Specific cell populations such as B cells, CD4 and CD8 T cells are also either isolated or characterized in conjunction with flow cytometry using cell-specific markers.


    Figure 1. Continuous and discontinuous Percoll gradient centrifugations

Representative data



Figure 2. A representative result from continuous Percoll gradient of TMEV-infected CNS cells

Flow cytometric analysis of CNS infiltrating cells isolated using continuous Percoll gradient at 7 days post infection is shown.

Notes

  1. A brief flow cytometry method is described below.
    1. After isolation of cells, Fc receptors were blocked using 50 μl of 2.4G2 hybridoma (ATCC) supernatant by incubation at 4 °C for 30 min.
    2. Cells were then stained with allophycocyanin-conjugated anti-CD8 (clone Ly-2) and PE-conjugated anti-CD4 (clone L3T4) at 4 °C for 30 min.
    3. To visualize macrophage and microglial populations, cells were stained with PE-conjugated anti-CD11b (clone M1/70) and allophycocyanin-conjugated anti-CD45 (clone Ly-5) antibodies. Antibodies diluted in Facs buffer (1% FBS, 0.09% sodium azide in PBS). After incubation, cells were washed with FACS buffer and resuspend the cells in 400 μl of FACS buffer.

Recipes

  1. Complete RPMI-1640 medium (100 ml)
    9 parts of RPMI and 1 part of fetal bovine serum
    Add 1 ml of 100x penicillin/streptomycin
  2. Collagenase and DNase I solution (100 ml)
    Mix 0.03 g Collagenase IV and 200 μl (10 mg/ml in PBS) DNase I into 100 ml HBSS
    Filter (0.22 μm) sterilize the solution
    These solutions are prepared fresh and kept at 4 °C for each use
  3. 100% Percoll (10 ml)
    Mix 1 ml 10x PBS and 9 ml Percoll
  4. 70% Percoll (100 ml)
    Mix 70 ml 100% Percoll and 30 ml PBS
  5. 30% Percoll
    Mix 30 ml 100% Percoll and 70 ml PBS

Acknowledgments

This protocol was adopted from many previous studies including the referenced work below and was supported by NIH (2RO1 NS28752 and NS33008) and NMSS (RG 4001A6 and RG 4342A7) grants.

References

  1. Havenith, C. E., Askew, D. and Walker, W. S. (1998). Mouse resident microglia: isolation and characterization of immunoregulatory properties with naive CD4+ and CD8+ T-cells. Glia 22(4): 348-359.
  2. Hou, W., Jin, Y. H., Kang, H. S. and Kim, B. S. (2014). Interleukin-6 (IL-6) and IL-17 synergistically promote viral persistence by inhibiting cellular apoptosis and cytotoxic T cell function. J Virol 88(15): 8479-8489.
  3. Jin, Y. H., Mohindru, M., Kang, M. H., Fuller, A. C., Kang, B., Gallo, D. and Kim, B. S. (2007). Differential virus replication, cytokine production, and antigen-presenting function by microglia from susceptible and resistant mice infected with Theiler's virus. J Virol 81(21): 11690-11702.

简介

与中枢神经系统(CNS)中的感染或炎症相关的免疫学和生物化学研究的多样性利用从初始和受影响的小鼠的CNS中分离的CNS驻留和/或浸润细胞,以研究潜在的机制和潜在的作用 的细胞群体。 将CNS细胞的机械和基于酶的单细胞制剂进行密度梯度以获得功能性单细胞。 与细胞特异性生物标志物组合,可以表征驻留小胶质细胞和浸润淋巴细胞(包括B和T细胞以及巨噬细胞)的功能和/或状态。

材料和试剂

  1. 异氟烷(Vedco,目录号:50201)
  2. Hanks平衡盐溶液(HBSS)(Cellgro,目录号:20-021-CV)
    注意:10x溶液,在无菌蒸馏水中稀释至1倍。
  3. RPMI-1640培养基(Sigma-Aldrich,目录号:R8758)
  4. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11055H)
  5. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140-122)
  6. 胶原酶IV型(Worthington Biochemical,目录号:4188)
  7. DNase I(Sigma-Aldrich,目录号:DN25)
  8. Percoll(GE Healthcare,目录号:17-0891-01)
  9. 磷酸盐缓冲盐水(PBS)
  10. 抗CD45-别藻蓝蛋白(APC)和抗-CD11b-藻红蛋白(PE)抗体(均来自BD,目录号分别为559864和557397)
  11. 抗CD8-APC(克隆Ly-; 2)(BD,目录号:553035)
  12. 抗CD4-PE(克隆L3T4)(BD,目录号:553049)抗体
  13. 完成RPMI-1640培养基(见配方)
  14. 胶原酶和DNA酶I溶液(参见配方)
  15. 100%Percoll(见配方)
  16. 70%Percoll(见配方)
  17. 30%Percoll(见配方)

设备

  1. 剪刀和镊子
  2. 10ml和50ml一次性塑料注射器(BD)
  3. 18 G针(BD)
  4. 钢网(250μm)(VWR International,目录号:AA13478-RR)
  5. 37°C水浴
  6. 台式冷冻离心机
  7. 高速离心机(Beckman Coulter,型号:J2-21)
  8. 50 ml螺旋盖管
  9. LSRII流式细胞仪(BD)
  10. 低速台式离心机(1,000-3,000 rpm)

程序

  1. 在感染后7天,通过异氟烷麻醉幼稚和病毒(Theiler's鼠脑脊髓炎病毒)感染的小鼠(分别为3-5对2-3)。
  2. 麻醉的小鼠用30ml无菌HBSS通过左心室使用50ml注射器经心脏灌注。
  3. 破坏小鼠和切除大脑。使用装有HBSS的10ml注射器从脊柱冲洗脊髓
  4. 使用附有橡胶的一次性注射器柱塞通过钢网将切除的大脑和脊髓单独研磨或一起研磨以制备单细胞悬浮液。
  5. 研磨的脑和脊髓经受300μg/ml IV型胶原酶加20μg/ml,并在37℃下孵育45分钟。
  6. 在室温下以630×g(1800rpm)旋转细胞悬浮液10分钟后,小心地倾析含胶原酶的上清液。

    使用连续渐变隔离CNS浸润细胞
    连续100%Percoll梯度用于富集CNS单核细胞。将10ml 100%Percoll加入到在完全RPMI中的20ml细胞悬浮液中,然后通过颠倒管子轻轻混合。使用J2-21离心机在25,000×g(15,000rpm)和37℃下30分钟不制动的Centifuge。通过吸出顶部15-20毫升中的细胞衰竭,收集底部三分之一的单核细胞。
  7. 转移细胞在底部Percoll梯度(10-15毫升)v底部50毫升管
  8. 用HBSS或RMPI使体积达到50ml,轻轻混合,然后在630×g(1800rpm)离心10分钟。
  9. 重悬细胞在完全RPMI培养基。
  10. 在TMEV感染后7天,从每只小鼠的脑和脊髓获得约1-2×10 6个细胞。从初始小鼠的CNS获得约2-5×10 5个细胞。

    使用不连续渐变隔离CNS浸润细胞
    或者,在70%和30%Percoll的界面处分离小胶质细胞和浸润性单核细胞。特别地,使用这种更确定的方法分离来自幼稚小鼠的小胶质细胞。如上所述(步骤1-6)制备的来自初始小鼠的3-5个脑和脊髓的单细胞悬浮液或来自感染小鼠的2-3个脑和脊髓经受不连续的Percoll梯度(70和30%)在2,800×g(5,000rpm)和 20℃20分钟(将细胞重悬于15ml 30%Percoll和底层20ml 70%Percoll中)。收集积聚在梯度界面中的细胞并转移到v-底50ml管中以进行洗涤。
  11. 将分离的CNS浸润单核细胞(5×10 5个)染色,并在基于FSC和SSC门控活细胞后通过流式细胞术分析(参见注释)。使用抗CD45-别藻蓝蛋白(APC)和抗-CD11b-藻红蛋白(PE)抗体来区分CNS驻留的CD45 - 小胶质细胞与浸润的CD45 - 小鼠胶质细胞,测定CD45和CD11b的表达。 /巨噬细胞的表达或其缺乏(淋巴细胞)。还使用细胞特异性标记物结合流式细胞术分离或表征特异性细胞群体例如B细胞,CD4和CD8T细胞。


    图1.连续和不连续的Percoll梯度离心

代表数据



图2. TMEV感染的CNS细胞的连续Percoll梯度的代表性结果

显示了在感染后7天使用连续Percoll梯度分离的CNS浸润细胞的流式细胞术分析。

笔记

  1. 下面描述简单的流式细胞术方法。
    1. 分离细胞后,使用50μl的2.4G2封闭Fc受体   杂交瘤(ATCC)上清液,通过在4℃下温育30分钟。
    2. 然后用别藻蓝蛋白缀合的抗CD8(克隆 Ly-2)和PE-缀合的抗CD4(克隆L3T4)在4℃下30分钟。
    3. 为了可视化巨噬细胞和小胶质细胞群,细胞 用PE-缀合的抗CD11b(克隆M1/70)染色 别藻蓝蛋白缀合的抗CD45(克隆Ly-5)抗体。 抗体   在Facs缓冲液(1%FBS,0.09%叠氮化钠的PBS溶液)中稀释。 后 孵育,用FACS缓冲液洗涤细胞并重悬细胞 在400μlFACS缓冲液中。

食谱

  1. 完成RPMI-1640培养基(100ml) 9份RPMI和1份胎牛血清 加入1ml 100x青霉素/链霉素
  2. 胶原酶和DNase I溶液(100ml) 将0.03g胶原酶IV和200μl(10mg/ml,在PBS中)将DNase I混合到100ml HBSS中
    过滤器(0.22μm)对溶液灭菌
    这些溶液是新鲜制备的,并且在每次使用时保持在4℃下
  3. 100%Percoll(10ml) 混合1ml 10x PBS和9ml Percoll
  4. 70%Percoll(100ml) 混合70ml 100%Percoll和30ml PBS
  5. 30%Percoll
    混合30ml 100%Percoll和70ml PBS

致谢

该协议从许多以前的研究中获得,包括下面参考的工作,并且由NIH(2RO1 NS28752和NS33008)和NMSS(RG 4001A6和RG 4342A7)授权支持。

参考文献

  1. Havenith,C.E.,Askew,D。和Walker,W.S。(1998)。 小鼠常驻小胶质细胞:用天然CD 4分离和表征免疫调节特性 >和CD 8 + T细胞。 22(4):348-359。
  2. Hou,W.,Jin,Y.H.,Kang,H.S.and Kim,B.S。(2014)。 白细胞介素-6(IL-6)和IL-17通过抑制细胞凋亡协同促进病毒的持久性细胞毒性T细胞功能。 J Virol 88(15):8479-8489。
  3. Jin,Y.H.,Mohindru,M.,Kang,M.H.,Fuller,A.C.,Kang,B.,Gallo,D.and Kim,B.S。(2007)。 差异病毒复制,细胞因子产生, 和由Theiler's病毒感染的易感和抗性小鼠的小胶质细胞的抗原呈递功能。 81(21):11690-11702。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jin, Y. and Kim, B. S. (2015). Isolation of CNS-infiltrating and Resident Microglial Cells. Bio-protocol 5(2): e1385. DOI: 10.21769/BioProtoc.1385.
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