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The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.

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Isolation and Purification of Murine Microglial Cells for Flow Cytometry
分离和纯化鼠小胶质细胞并进行流式细胞术

神经科学 > 细胞机理 > 细胞分离和培养
作者: Peter Theriault
Peter TheriaultAffiliation: Neuroscience Laboratory, CHU de Quebec Research Center and department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Canada
Bio-protocol author page: a2850
Maude Bordeleau
Maude BordeleauAffiliation: Neuroscience Laboratory, CHU de Quebec Research Center and department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Canada
Bio-protocol author page: a2851
 and Serge Rivest
Serge RivestAffiliation: Neuroscience Laboratory, CHU de Quebec Research Center and department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Canada
For correspondence: serge.rivest@crchudequebec.ulaval.ca
Bio-protocol author page: a2849
Vol 6, Iss 1, 1/5/2016, 1981 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1703

[Abstract] The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.
Keywords: Flow Cytometry(流式细胞仪), Microglial cell(小胶质细胞), Mouse(鼠标), Brain(脑)

[Abstract]

Materials and Reagents

  1. Syringe 10 ml (BD bioscience, catalog number: 309604 )
  2. Needle 22 G (BD bioscience, catalog number: 305155 )
  3. Syringe 1 ml 27 G (BD bioscience, catalog number: 309623 )
  4. 15 ml centrifugation tube (Thermo Fisher Scientific, catalog number: 339651 )
  5. 50 ml centrifugation tube (Thermo Fisher Scientific, catalog number: 339653 )
  6. 96 v-shaped wells plate (Thermo Fisher Scientific, catalog number: 249662 )
  7. 5 ml polystyrene FACS tube (BD Biosciences, Falcon®, catalog number: 352054 )
  8. 70 µm nylon filter (VWR International, catalog number: 352350 )
  9. Mice
  10. PBS without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-010-CL )
  11. Ice cold Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2+/Mg2+/ Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
  12. PBS 10x without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-012-CL )
  13. Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) (Sigma-Aldrich, catalog number: T7254 )
  14. Liberase TL Research Grade (Roche Diagnostics, catalog number: 05401020001 )
  15. DNAse1 (Roche Diagnostics, catalog number: 11284932001 )
  16. HEPES (1 M) (Sigma-Aldrich, catalog number: H0887 )
  17. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F1051 )
  18. EDTA (Sigma-Aldrich, catalog number: E16144 )
  19. Percoll (GE Healthcare, Dharmacon, catalog number: 17-0891-01 )
  20. HBSS 10x without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-506-CL )
  21. HBSS without Ca2+/Mg2+ with red phenol (Life technologies, catalog number: 14170-112 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14170-112”.
  22. Purified Rat anti-mouse CD16/CD32 (BD Bioscience, catalog number: 553141 )
  23. Anti-mouse CD45 PE/Cy5 (BD Bioscience, catalog number: 553082 )
  24. Anti-mouse CD11b Alexa 700 (eBioscience, catalog number: 56-0112-82 )
  25. Anti-mouse CD16/CD32 (1:100) (BD Biosciences, catalog number: 553142 )
  26. Live/Dead Fixable Blue Dead Cell (Life technologies, catalog number: L23105 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L23105”.
  27. Digestion medium (see Recipes)
  28. Ice-cold FACS buffer (see Recipes)
  29. Percoll dilution medium (see Recipes)
  30. PFA 4% (w/v) solution (see Recipes)

Equipment

  1. Tissue grinder Capacity 3 ml (VWR International, catalog number: 886000-0020 )
  2. Centrifuge Sorvall legend RT (Mandel, catalog number: 75004377 )
  3. Flow cytometer BD Canto II or LSR II (BD bioscience)
  4. Peristaltic pump, facultative (Cole-Parmer Instrument Company, catalog number: EW-78001-02 )

Software

  1. BD FACS Diva software (version 6.1.2)

Procedure

  1. Stage 1: Animal preparation and perfusion
    1. Anesthetize mice via an i.p. injection of a ketamine hydrochloride and xylazine mixture (10 ml and 1 ml respectively).
    2. Flush deeply anesthetized mice with ice-cold sterile DPBS (perfuse transcardially with cold dPBS using peristaltic pump or a 10 ml syringe connect with 22G needle. Many well describe protocols are available online but for good examples see References Video 1 and Video 2.

  2. Stage 2: Brain extraction and Isolation of microglial cells
    1. Extract one brain and put the brain tissues into a tissue grinder containing 2 ml of ice-cold DPBS without Ca2+/Mg2+.
    2. After homogenization, transfer homogenates into 15 ml centrifugation tubes (homogenize the tissues completely by doing up and down with the grinder bar).
    3. Rinse three times the plunger with 2 ml of ice-cold DPBS. Keep on ice.
    4. Centrifuge homogenates at 300 x g for 10 min at 4 °C.
    5. Aspirate supernatants and gently resuspend pellets in 3 ml of Digestion medium.
    6. Incubate homogenates at 37 °C for 40 min and shake tubes every 10 min.
    7. After digestion, complete volumes to 10 ml with ice-cold FACS buffer.
    8. Transfer homogenates into new 50 ml centrifugation tubes by filtering cell suspensions through 70 µm nylon filters.
    9. Wash cells by rinsing the 15 ml centrifugation tube with 10 ml of ice-cold FACS buffer and transfer into the 50 ml centrifugation tube through the same 70 µm-filter.
    10. Centrifuge homogenates at 300 x g for 10 min at 4 °C.
    11. Aspirate supernatants and wash cells by adding 20 ml of ice-cold FACS buffer.
    12. Centrifuge homogenates once again at 300 x g for 10 min at 4 °C.
    13. During the centrifugations, prepare the isotonic Percoll solution by diluting Percoll 10:1 in 10x HBSS. Prepare needed volumes of 30% and 80% Percoll solutions by diluting the Percoll isotonic solution with the Percoll dilution medium.
      Note: Keep Percoll solutions at room temperature.
    14. Resuspend cell pellets in 8 ml of 30% Percoll solution.
    15. Transfer 4 ml into a 15 ml centrifugation tube.
    16. Using a serological pipette, carefully transfer 3 ml of 80% Percoll solution below the cell containing 4 ml of 30% Percoll solution.
      Note: Take 4-5 ml in the serological pipette and use gravity to underlay the 80% Percoll solution to avoid the mixture of the upper and lower layers, which could affect the efficiency and yield of microglia isolation.
    17. Add the remaining cell containing 30% Percoll solution to the upper phase.
    18. Centrifuge at 1,050 x g with no brakes and low acceleration for 40 min at room temperature.
    19. Aspirate the fat containing layer at the top of the liquid.
    20. Using a P1000 micropipette, carefully collect isolated microglial cells (~2 ml) into a new 15 ml centrifugation tube containing 10 ml of ice-cold FACS buffer.
      Note: Microglial cells are within the cell ring at the interface between the pink top layer (30% Percoll) and the colorless bottom layer (80% Percoll).
    21. Centrifuge isolated microglial cells at 300 x g for 10 min at room temperature.
    22. Aspirate supernatants and resuspend pelleted cells into 250 µl of ice-cold FACS Buffer.


      Figure 1. Schematic diagram of the gradient preparation

  3. Stage 3: Microglia cell markers staining and flow cytometry
    1. Transfer all cell suspensions (250 µl) into 96 v-shaped well plate.
    2. Centrifuge cells at 300 x g for 3 min at 4 °C and remove supernatants by inverting the plate.
    3. Resuspend cells into 200 µl of ice-cold FACS buffer containing purified rat anti-mouse CD16/CD32 (1:100) for Fc receptors blocking.
    4. Incubate on ice for 20 min.
    5. Centrifuge cells at 300 x g for 3 min at 4 °C and remove supernatants.
    6. Resuspend cells into 50 µl of ice-cold FACS buffer containing rat anti-mouse CD45 PEG/Cy5 (1:50), rat anti-mouse CD11b Alexa 700 (1:50) and Live/Dead Blue fluorescent reactive dye (1:50).
    7. Incubate on ice for 45 min.
    8. Add 200 µl of ice-cold FACS buffer.
      Note: At this step, it is possible to fix cells for a subsequent analysis on the flow cytometer by adding 50 µl of 4% (w/v) paraformaldehyde (PFA) solution at pH = 7.4. Incubate at room temperature for 15 min and wash cells with ice-cold FACS buffer prior to their storage at 4 °C in the dark, for up to 4 days.
    9. Centrifuges cells at 300 x g for 3 min at 4 °C.
    10. Resuspend cells into 250 µl of ice-cold FACS buffer and transfer into 5 ml polystyrene tubes for flow cytometry.
    11. Analyze samples by using a two-laser, six-colors FACS Canto II flow cytometer and data acquisition with BD FACSDiva software (version 6.1.2).

Representative data


Figure 2. Example of results following an analysis of isolated microglial cells by flow cytometry. Dot plot showing a representative sample of isolated microglial cells analyzed by flow cytometry following a Live/Dead-Blue, CD45-PE-Cy5 and CD11b-AF700 immunofluorescent staining.

Recipes

  1. Digestion medium
    TLCK 0.5 µg/ml
    DNAse1 25 ng/ml
    Liberase 8.125 pg/ml
    HEPES 20 mM
    In sterile water
  2. Ice-cold FACS buffer
    5% FBS, EDTA (20 mM), HEPES (2 mM) dilute in PBS
  3. Percoll dilution medium
    Red phenol HBSS with EDTA (2 mM) and HEPES (20 mM)
  4. PFA 4% (w/v) solution (pH = 7.4)
    NaOH (50 mN) 4% (w/v) paraformaldehyde
    50 mN NaOH
    26.5 mM NaH2PO4
    77 mM Na2HPO4
    (77 mM)

Acknowledgments

This work was supported by grants from the Canadian Institutes for Health Research (CIHR) and the Multiple Sclerosis Scientific Research Foundation of Canada. This protocol was adapted from previous work of Martine Lessard and Antoine Lampron.

References

  1. Lampron, A., Larochelle, A., Laflamme, N., Prefontaine, P., Plante, M. M., Sanchez, M. G., Yong, V. W., Stys, P. K., Tremblay, M. E. and Rivest, S. (2015). Inefficient clearance of myelin debris by microglia impairs remyelinating processes. J Exp Med 212(4): 481-495.
  2. Video 1: www.youtube.com/watch?v=szvmT5iI0z0.
  3. Video 2: www.youtube.com/watch?v=3H8K7ooPWLs.

材料和试剂

  1. 注射器10ml(BD bioscience,目录号:309604)
  2. Needle 22G(BD bioscience,目录号:305155)
  3. 注射器1ml 27G(BD bioscience,目录号:309623)
  4. 15ml离心管(Thermo Fisher Scientific,目录号:339651)
  5. 50ml离心管(Thermo Fisher Scientific,目录号:339653)
  6. 96孔V型孔板(Thermo Fisher Scientific,目录号:249662)
  7. 5ml聚苯乙烯FACS管(BD Biosciences,Falcon ,目录号:352054)
  8. 70μm尼龙过滤器(VWR International,目录号:352350)
  9. 小鼠
  10. PBS(不含Ca 2+ 2 + /Mg 2 + (BOLLéCOMMUNICATIONS,Wisent,目录号:311-010-CL)
  11. 不含Ca 2+/Mg 2+/Dulbecco磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D8537)的冰冷Dulbecco's磷酸盐缓冲盐水(DPBS)
  12. PBS 10x,不含Ca 2+ 2 + /Mg 2 + (BOLLéCOMMUNICATIONS,Wisent,目录号:311-012-CL)
  13. Nα-甲苯磺酰基-L-赖氨酸氯甲基酮盐酸盐(TLCK)(Sigma-Aldrich,目录号:T7254)
  14. Liberase TL研究级(Roche Diagnostics,目录号:05401020001)
  15. DNAse1(Roche Diagnostics,目录号:11284932001)
  16. HEPES(1M)(Sigma-Aldrich,目录号:H0887)
  17. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F1051)
  18. EDTA(Sigma-Aldrich,目录号:E16144)
  19. Percoll(GE Healthcare,Dharmacon,目录号:17-0891-01)
  20. (BOLLéCOMMUNICATIONS,Wisent,目录号:311-506-CL)的HBSS 10x,其中含有Ca 2+ 2+ /Mg 2 +
  21. (Life technologies,目录号:14170-112)的HBSS,不含Ca 2+/Mg 2+/H 2 SO 4 + 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14170-112"。
  22. 纯化的大鼠抗小鼠CD16/CD32(BD Bioscience,目录号:553141)
  23. 抗小鼠CD45 PE/Cy5(BD Bioscience,目录号:553082)
  24. 抗小鼠CD11b Alexa 700(eBioscience,目录号:56-0112-82)
  25. 抗小鼠CD16/CD32(1:100)(BD Biosciences,目录号:553142)
  26. 活/死可固定蓝色死细胞(生命技术,目录号:L23105)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:L23105" br />
  27. 消化介质(参见配方)
  28. 冰冷的FACS缓冲液(参见配方)
  29. Percoll稀释介质(参见配方)
  30. PFA 4%(w/v)溶液(参见配方)

设备

  1. 组织研磨机容量3ml(VWR International,目录号:886000-0020)
  2. 离心Sorvall图例RT(Mandel,目录号:75004377)
  3. 流式细胞仪BD Canto II或LSR II(BD bioscience)
  4. 蠕动泵,兼性(Cole-Parmer Instrument Company,目录号:EW-78001-02)

软件

  1. BD FACS Diva软件(版本6.1.2)

程序

  1. 阶段1:动物准备和灌注
    1. 通过腹腔麻醉小鼠。注射氯胺酮盐酸盐和甲苯噻嗪混合物(分别为10ml和1ml)
    2. 用冰冷的无菌DPBS(灌注。)冲洗深度麻醉的小鼠 用冷的dPBS使用蠕动泵或10ml注射器经心脏 连接22G针。许多很好描述的协议可用 在线,但好的例子见参考文献视频1和视频2。

  2. 阶段2:脑提取和小胶质细胞的分离
    1. 提取一个脑,并将脑组织放入含有2ml没有Ca 2+ 2 + /Mg 2 + 的冰冷DPBS的组织研磨机中。
    2. 匀浆后,将匀浆转移至15ml离心 ?管(通过与上下同时完成组织匀浆) 研磨棒)。
    3. 用2ml冰冷的DPBS冲洗柱塞三次。保持冰上。
    4. 在4℃下以300×g离心匀浆10分钟。
    5. 吸出上清液,轻轻地将沉淀重悬于3ml消化培养基中
    6. 孵育匀浆在37℃下40分钟,摇动管每10分钟
    7. 消化后,用冰冷的FACS缓冲液将体积完全加至10ml
    8. 通过70微米尼龙过滤器过滤细胞悬浮液,将匀浆转移到新的50ml离心管中
    9. 洗涤细胞通过冲洗15毫升离心管用10毫升 冰冷的FACS缓冲液,并转移到50ml离心管中 通过相同的70μm过滤器
    10. 在4℃下以300×g离心匀浆10分钟。
    11. 吸出上清液,加入20ml冰冷的FACS缓冲液洗涤细胞
    12. 在4℃下再次离心匀浆,在300×g离心10分钟
    13. 在离心期间,制备等渗的Percoll溶液 通过在10x HBSS中稀释Percoll 10:1。准备所需体积的30%和 80%Percoll溶液,用Percoll等渗溶液稀释 ?Percoll稀释介质。
      注意:保持Percoll溶液在室温下。
    14. 将细胞沉淀重悬于8ml 30%Percoll溶液中
    15. 将4 ml转移到15 ml离心管中。
    16. 使用血清移液管,小心转移3毫升80%Percoll ?溶液在含有4ml 30%Percoll溶液的细胞下面 注意:在血清学移液管中取4-5 ml,并使用重力作用于底层 ?80%Percoll溶液避免上下混合 层,这可能影响小胶质细胞的效率和产量 隔离。
    17. 将剩余的含有30%Percoll溶液的细胞加入上层相
    18. 以1,050 x g 离心,无制动器,在室温下低加速40分钟。
    19. 吸出液体顶部的含脂肪层。
    20. 使用P1000微量移液管,仔细收集分离的小胶质 细胞(约2ml)转移到含有10ml的新的15ml离心管中 冰冷的FACS缓冲液 注意:小胶质细胞在细胞内 环在粉红色顶层(30%Percoll)和 无色底层(80%Percoll)。
    21. 在室温下将分离的小胶质细胞在300×g离心10分钟
    22. 吸出上清液,并将沉淀的细胞重悬在250μl冰冷的FACS缓冲液中。


      图1.梯度准备的示意图

  3. 阶段3:小神经胶质细胞标记染色和流式细胞术
    1. 将所有细胞悬浮液(250μl)转移到96v形孔板中
    2. 在4℃下以300×g离心细胞3分钟,并通过倒置平板除去上清液。
    3. 将细胞重悬于200μl冰冷的FACS缓冲液中 纯化的大鼠抗小鼠CD16/CD32(1:100)用于Fc受体阻断
    4. 在冰上孵育20分钟。
    5. 在4℃下以300×g离心细胞3分钟,除去上清液。
    6. 将细胞重悬在50μl含有大鼠的冰冷的FACS缓冲液中 抗小鼠CD45 PEG/Cy5(1:50),大鼠抗小鼠CD11b Alexa 700(1:50) 和活/死蓝荧光活性染料(1:50)
    7. 在冰上孵育45分钟。
    8. 加入200μl冰冷的FACS缓冲液 注意:在这一步,可以修复后续的单元格 在流式细胞仪上通过加入50μl的4%(w/v) 多聚甲醛(PFA)溶液。在室温下孵育 温度15分钟,然后用冰冷的FACS缓冲液洗涤细胞 ?它们在4℃下在黑暗中储存最多4天。
    9. 在4℃下以300×g离心细胞3分钟。
    10. 将细胞重悬在250μl冰冷的FACS缓冲液中,并转移到5ml聚苯乙烯管中进行流式细胞术。
    11. 使用双激光六色FACS Canto II流分析样品 ?细胞仪和数据采集与BD FACSDiva软件(版本 6.1.2)。

代表数据


图2.通过流式细胞术分析分离的小胶质细胞后的结果的实施例点图显示了在活/死蓝,CD45-PE-标记后,通过流式细胞术分析的分离的小胶质细胞的代表性样品。 Cy5和CD11b-AF700免疫荧光染色。

食谱

  1. 消化介质
    TLCK0.5μg/ml
    DNAse1 25ng/ml
    Liberase 8.125pg/ml
    HEPES 20mM
    在无菌水中
  2. 冰冷的FACS缓冲区
    5%FBS,EDTA(20mM),HEPES(2mM)稀释于PBS中
  3. Percoll稀释介质
    红色酚HBSS与EDTA(2mM)和HEPES(20mM)
  4. PFA 4%(w/v)溶液(pH = 7.4) NaOH(50mN)4%(w/v)多聚甲醛
    50 mN NaOH
    26.5mM NaH 2 PO 4 4/v/v 77mM Na 2 HPO 4
    (77mM)

致谢

这项工作得到加拿大健康研究所(CIHR)和加拿大多发性硬化症科学研究基金会的资助。该协议改编自Martine Lessard和Antoine Lampron的以前的工作。

参考文献

  1. Lampron,A.,Larochelle,A.,Laflamme,N.,Prefontaine,P.,Plante,M.M.,Sanchez,M.G.,Yong,V.W.,Stys,P.K.,Tremblay,M.E.and Rivest, 小胶质细胞对髓磷脂碎片的无效清除损害了髓鞘再生过程。 J Exp Med 212(4):481-495。
  2. 视频1: www.youtube.com/watch?v=szvmT5iI0z0
  3. 视频2: www.youtube.com/watch?v=3H8K7ooPWLs
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How to cite this protocol: Theriault, P., Bordeleau, M. and Rivest, S. (2016). Isolation and Purification of Murine Microglial Cells for Flow Cytometry. Bio-protocol 6(1): e1703. DOI: 10.21769/BioProtoc.1703; Full Text



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