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Isolation, Purification, and Culture of Primary Murine Microglia Cells
原代小鼠小胶质细胞的分离、纯化和培养   

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Abstract

The following is a detailed protocol for the isolation, purification and culture of murine brain microglia cells using neutral enzyme digestion and shaking. The protocol below is designed to isolate and culture a large number of purified inactivated microglia cells. Neutral enzyme digestion allows for minimal cellular damage and a higher cell recovery compared to other mechanical dissociative methods such as mincing. Cells cultured using this method will display the size and morphological features of microglia cells.

Keywords: Isolation(隔离), Purification(净化), Primary(初级的), Microglia(小胶质细胞)

Materials and Reagents

  1. Five to eight fetal (1-3 days old) BALB/C mice (Harlan)
  2. Ice
  3. DMEM (Sigma-Aldrich, catalog number: D5796 )
  4. Fetal bovine serum heat inactivated (FBS) (Sigma-Aldrich, catalog number: F9665 )
  5. D-Hank’s balanced salt solution without phenol red (Life Technologies, InvitrogenTM, catalog number: H6648 )
  6. Trypsin / EDTA (0.05%/0.02%) (Life Technologies, InvitrogenTM, catalog number: 25300054 )
  7. Alcohol (70%)
  8. Poly-D-lysine solution (10 μg/ml) (Sigma-Aldrich, catalog number: L202 )
  9. 70% Alcohol
  10. Physiological saline (0.9%) (Sigma-Aldrich, catalog number: 07982-100TAB-F )
  11. Trypsin (0.25%) (Life Technologies, InvitrogenTM, catalog number: 25200-072 )
    Two different concentrations of DMEM with FBS are used in order to maximize growth. The logic behind this is that the micrglia are more ‘sensitive’ after enzymic digestion. Therefore the use of DMEM with a higher concentration of FBS (i.e. 15%) provides additional growth factors and buffering agents after disaggregation and its use prior to enzymic expose is to allow the cells to acclimatize to this higher concentration and to optimize the cells under this improved growing condition. DMEM +FBS 10% is sufficient for culturing at all other periods.

Equipment

  1. Straight scissors
  2. Curved scissors
  3. Corneal scissors
  4. Straight forceps (small, x2)
  5. Curved artery forceps (x3)
  6. Beaker (100 ml, x2)
  7. Culture dish (30 mm, x2)
  8. Culture flask (25 cm2 and 75 cm2)
  9. Centrifuge Falcon tubes (15 ml and 50 ml)
  10. Serological pipettes (2 ml and 5 ml)
  11. Plastic transfer pipettes (3.5 ml)
  12. 70-μm nylon cell strainer
  13. Sterile gauze
  14. Incubator (37 °C; 5% CO2; humidified)
  15. Refrigerated centrifuge
  16. Magnifying glass and microscope
  17. UV light
  18. Culture flask with mixed glia cells
  19. Parafilm
  20. Alcohol burner
  21. Orbital shaker/Belly dancer (37 °C)
  22. 24-well tissue culture plate pre-coated with poly-D-lysine

Procedure

Caution: Bio-safety practices (Level II) should be adhered to during handling of both animals and cell cultures.
Note: All procedures are performed under aseptic conditions.

  1. Stage 1: Isolation and culture of murine microglia cells via enzymatic digestion.
    Enzymatic disaggregation of mouse brain tissue with trypsin provides a high yield of microglia cells without any detectable cell activations.
    1. The base of a 75 cm2 culture flask is coated with 5 ml poly-D-lysine (10 μg/ml) and allowed to set overnight at room temperature (note: Poly-D-lysine assists in the cells adhering to the flask).
    2. On the day of the experiment all excess poly-D-lysine is removed as any excess solution is caustic to the microglia cells. The flask is then washed once with PBS. In addition the cleaned culture cabinet/hood is exposed to an additional 30 min of UV light to maximize the sterilization process.
    3. Five to eight fetal BALB/c mice (1-3 days old) are culled by cutting both carotid arteries (without decapitating the mouse; otherwise subsequent handling is made difficult) with a pair of scissors and the heads are placed in a beaker with 70% alcohol for 15-30 sec. Step (3) to (g) is represented by Figure 1 (see below).
    4. The decapitated heads are removed and all excess blood and alcohol is removed using a piece of sterile gauze.
    5. Next the skin over the skull is cleared under aseptic technique. This is achieved by making a crucifix incision over the cranium (transverse incision at the base of the skull and the vertical incision along the midline of the skull up to the anterior part of the skull). A new straight forceps is then used to peel away the skin on the scalp to fully expose the skull.
    6. The lambda and sagittal sutures are identified. A pair of corneal scissors are used to cut along the suture lines. A 3rd pair of straight forceps are then used to ‘peel’ the skull away (note: The skull of a mouse this age is very soft and pliable) and to fully expose the brain (note: The following 2 steps must be performed on ice to minimize cell loss).
    7. A new curved forceps is then inserted under the base of the brain (taking care not to damage the brain). Gently lift the brain away from the skull base. Place the brain onto a small culture dish that contains a small volume of D-Hanks balanced salt solution.
    8. The meningeal lining (dura and arachnoid layers) is then gently removed using two small straight forceps under a magnifying glass. The cleaned brain is then placed in a new culture dish with 2 ml of trypsin / EDTA (0.05%/0.02%). The brain is then minced into a fine slurry using a pair of corneal scissors.
    9. The brain / trypsin suspension is transferred gently into a 15 ml centrifuge tube using a 5 ml serological pipette and the same volume of trypsin / EDTA (0.05%/0.02%) is added. The tube is then maintained in the incubator at 37 °C in a humidified 5% CO2 atmosphere for 8-10 min to allow for enzymatic disaggregation.
    10. After the allocated time, DMEM with 10% FBS is added to the suspension (1:1 ratio) to neutralize the trypsin. The single cell suspension is gently agitated by moving the solution up and down 20 times using a 3.5 ml plastic transfer pipette with care being taken to avoid bubble formation.
    11. The suspension is allowed to stand for a minute at room temperature to allow any large pieces to precipitate. This supernatant is passed through a 70 μm nylon cell strainer into another 100 ml beaker. The process of agitation using a 3.5 ml plastic transfer pipette with more DMEM with 10% FBS (equal volume to the supernatant) and straining is repeated twice.
    12. All the filtrated fluid is then gently transferred into a 50 ml centrifuge tube using a serological pipette. This is then centrifuged in a cooled (4 °C) centrifuge at 190 x g for 8 min.
    13. The supernatant is then discarded and the cell pellet is resuspended in 15 ml of DMEM containing 15% FBS.
    14. The resuspended cells are added to the poly-D-lysine coated flask and incubated at 37 °C in a humidified 5% CO2 atmosphere. After 3 days, the DMEM is replaced with a fresh batch of 15 ml of DMEM containing 10% FBS. After this, the medium is changed every alternate day using a 1:1 mix of fresh DMEM containing 10% FBS and the supernatant of the medium of the flask in order to remove any dead cells and retain existing growth factors previously released by the cells. This is achieved by removing the existing 15 ml via a serological pipette into a 50 ml centrifuge tube period followed by an immediate replacement with 7.5 ml of fresh DMEM with 10% FBS. The ‘old’ medium is then centrifuged to remove dead cells at 190 x g. To make the 15 ml required for the flask, 7.5 ml of the centrifuged supernatant is added. This process can be repeated for up to 10-14 days.
    15. When the cells are approximately 80-90% confluent, they can be used for purification.

  2. Stage 2: Purification of murine microglia cells from the mixed glia cell culture
    1. Once the mixed glia cell culture achieves a 90% confluence level (after 10-14 days) the microglia cells can be detached from the flask using the heated orbital shaker/belly dancer machine at 240 rpm for 1.5 - 2 h (37 °C). Twenty-hour hours prior to disaggregation, the medium is replaced with fresh DMEM containing 15% FBS.
    2. The next day the flask is placed on the heated orbital shaker at 200-240 rpm/min at 37 °C for 2 h.
    3. The suspension is transferred into a 50 ml centrifuge tube and then centrifuged at 190 x g for 8 min.
    4. The supernatant is discarded and the cell pellet is resuspended in fresh DMEM containing 15% FBS.
    5. The cells are then seeded onto a 24-well tissue culture plate that has been pre-coated with 5 ml of poly-D-lysine (10 μg/ml). The cells are distributed into 7.5 x 104 cells/well and incubated at 37 °C in a humidified 5% CO2 atmosphere. The medium is replaced every 2-3 days with fresh DMEM containing 10% FBS. Cell growth is observed regularly using a microscope (Figure 2). The cellular yield is 1 x 106 microglia / mouse.
    6. The original mixed glia cell flask is replenished with 10 ml fresh DMEM containing 10% FBS. After this, the medium is changed every 2-3 days using a 1:1 mix of fresh DMEM containing 10% FBS and the supernatant of the medium of the flask as described above. The purification process described above can then be repeated a week later. After plating the cells in the 24-well plate, the cells must be used within a week (optimal for the first 3 days). The cell purity can be assessed via flow cytometry using the anti-CD11b monoclonal antibody.


      Figure 1.  Method for the removal of the brain from a 1-3 day old mouse


      Figure 2. Microglia on light microscopy

Acknowledgments

This work was supported by National Natural Science Foundation of China (grants 30972718 and 81273242), Natural Science Foundation of Jiangsu Province (grant BK2012605), and Jiangsu Province Program of Innovative and Entrepreneurial Talents (2011-2014).

References

  1. Chen, X., Quinn, E. M., Ni, H., Wang, J., Blankson, S., Redmond, H. P., Wang, J. H. and Feng, X. (2012). B7-H3 participates in the development of experimental pneumococcal meningitis by augmentation of the inflammatory response via a TLR2-dependent mechanism. J Immunol 189(1): 347-355.

简介

以下是使用中性酶消化和摇动的鼠脑小胶质细胞的分离,纯化和培养的详细方案。 以下方案设计用于分离和培养大量纯化的灭活的小神经胶质细胞。 与其他机械解离方法例如切碎相比,中性酶消化允许最小的细胞损伤和更高的细胞回收率。 使用这种方法培养的细胞将显示小神经胶质细胞的大小和形态特征。

关键字:隔离, 净化, 初级的, 小胶质细胞

材料和试剂

  1. 5-8只胎儿(1-3天龄)BALB/C小鼠(Harlan)
  2. 冰块
  3. DMEM(Sigma-Aldrich,目录号:D5796)
  4. 胎牛血清热灭活(FBS)(Sigma-Aldrich,目录号:F9665)
  5. 不含酚红的D-Hank平衡盐溶液(Life Technologies,Invitrogen TM ,目录号:H6648)
  6. 胰蛋白酶/EDTA(0.05%/0.02%)(Life Technologies,Invitrogen TM ,目录号:25300054)
  7. 酒精(70%)
  8. 聚D-赖氨酸溶液(10μg/ml)(Sigma-Aldrich,目录号:L202)
  9. 70%酒精
  10. 生理盐水(0.9%)(Sigma-Aldrich,目录号:07982-100TAB-F)
  11. 胰蛋白酶(0.25%)(Life Technologies,Invitrogen TM ,目录号:25200-072)
    使用两种不同浓度的具有FBS的DMEM以使生长最大化。 这背后的逻辑是微胶粒在酶消化后更"敏感"。 因此,具有更高浓度的FBS(即15%)的DMEM的使用在解聚之后提供另外的生长因子和缓冲剂,并且在酶暴露之前其使用是允许细胞适应这种更高的浓度 并优化该细改善生长条件。 DMEM + FBS 10%足以在所有其他时期进行培养。

设备

  1. 直剪
  2. 弯曲剪刀
  3. 角膜剪刀
  4. 直钳(小,x2)
  5. 弯曲动脉钳(×3)
  6. 烧杯(100ml,x2)
  7. 培养皿(30mm,x2)
  8. 培养瓶(25cm 2和75cm 2)
  9. 离心Falcon管(15ml和50ml)
  10. 血清移液管(2 ml和5 ml)
  11. 塑料移液管(3.5 ml)
  12. 70-μm尼龙细胞过滤器
  13. 无菌纱布
  14. 培养箱(37℃; 5%CO 2;加湿)
  15. 冷冻离心机
  16. 放大镜和显微镜
  17. 紫外线
  18. 具有混合胶质细胞的培养瓶
  19. parafilm
  20. 酒精燃烧器
  21. 冷冻离心机
  22. 轨道摇床/肚皮舞(37℃)
  23. 预先用聚-D-赖氨酸包被的24孔组织培养板

程序

注意:在处理动物和细胞培养物时应遵守生物安全实践(II级)。
注意:所有程序均在无菌条件下进行。

  1. 阶段1:通过酶消化分离和培养鼠小神经胶质细胞 小鼠脑组织与胰蛋白酶的酶解聚合提供了高产率的小胶质细胞,没有任何可检测的细胞活化。
    1. 用5ml聚-D-赖氨酸(10μg/ml)包被75cm 2培养瓶的底部,并使其在室温下放置过夜(注意:Poly-D- 赖氨酸帮助细胞粘附于烧瓶上)。
    2. 在实验当天,除去任何过量的聚-D-赖氨酸,因为任何过量的溶液对小胶质细胞都是苛性的。 然后将烧瓶用PBS洗涤一次。 另外清洁的文化 橱柜/罩子暴露于额外的30分钟的UV光,以最大化灭菌过程。
    3. 通过用一把剪刀切割两个颈动脉(不使小鼠断头,否则难以进行随后的处理)来挑取5至8只胎儿BALB/c小鼠(1-3天龄),将头部置于具有70 %酒精15-30秒。步骤(3)至(g)由图1(见下文)表示。
    4. 去掉头盖,用一片无菌纱布除去所有过量的血液和酒精。
    5. 接下来,在无菌技术下清除颅骨上的皮肤。这是通过在颅骨上的十字架切口(在头骨的基部的横向切口和沿着颅骨的中线的垂直切口直到颅骨的前部)实现的。然后使用新的直镊子剥离在头皮上的皮肤以完全暴露颅骨。
    6. 识别λ和矢状缝合线。使用一对角膜剪刀沿着缝合线切割。然后使用一对3直的镊子来"剥离"头骨(注意:这个年龄的老鼠的头骨是非常柔软和柔韧的),并且完全暴露大脑(n°ote:必须在冰上进行以下2个步骤以最小化细胞丢失)。
    7. 然后将新的弯曲镊子插入大脑底部(注意不要损伤大脑)。轻轻地抬起大脑远离骷髅基地。将大脑放在一个小的文化菜 含有少量D-Hanks平衡盐溶液。
    8. 然后在放大镜下使用两个小直镊子轻轻地移除脑膜内层(硬脑膜和蛛网膜层)。然后将清洁的脑置于具有2ml胰蛋白酶/EDTA(0.05%/0.02%)的新培养皿中。然后使用一对角膜剪刀将大脑切成细浆。
    9. 使用5ml血清移液管将脑/胰蛋白酶悬浮液轻轻转移到15ml离心管中,并加入相同体积的胰蛋白酶/EDTA(0.05%/0.02%)。然后将管在37℃下在湿润的5%CO 2气氛中在培养箱中保持8-10分钟以允许酶促解聚。
    10. 在分配的时间后,将具有10%FBS的DMEM加入到悬浮液(1:1比例)中以中和胰蛋白酶。通过使用3.5ml塑料转移移液管上下移动溶液20分钟轻轻搅动单细胞悬浮液,小心避免形成气泡。
    11. 将悬浮液在室温下静置一分钟以使任何大块沉淀。将该上清液通过70μm尼龙细胞过滤器进入另一个100ml烧杯中。使用具有更多具有10%FBS(等于上清液的体积)的DMEM和应变的3.5ml塑料转移移液管的搅拌过程重复两次。
    12. 然后使用血清移液管将所有过滤的流体轻轻转移到50ml离心管中。然后将其在冷却(4℃)的离心机中在190×g离心8分钟。
    13. 然后弃去上清液,将细胞沉淀重悬于15ml含有15%FBS的DMEM中
    14. 将重悬浮的细胞加入到聚-D-赖氨酸包被的烧瓶中并在37℃下在潮湿的5%CO 2气氛中温育。 3天后,用新鲜批次的15ml含有10%FBS的DMEM替换DMEM。此后,每隔一天使用含有10%FBS的新鲜DMEM和烧瓶培养基的上清液的1:1混合物更换培养基,以除去任何死细胞并保留细胞先前释放的现有生长因子。这通过将现有的15ml通过血清移液管移入50ml离心管期,然后立即用7.5ml具有10%FBS的新鲜DMEM置换来实现。然后将该"旧"培养基在190×g离心以去除死细胞。为了制备烧瓶所需的15ml,加入7.5ml离心的上清液。该过程可重复长达10-14天。
    15. 当细胞约80-90%汇合时,它们可以用于纯化
  2. 阶段2:从混合的神经胶质细胞培养物中纯化鼠小神经胶质细胞
    1. 一旦混合的神经胶质细胞培养物达到90%汇合水平(在10-14天后),可以使用加热的轨道摇床/肚皮舞蹈机在240rpm下将小神经胶质细胞从烧瓶中分离1.5-2小时(37℃) 。在解聚之前二十小时,用含有15%FBS的新鲜DMEM替换培养基。
    2. 第二天,将烧瓶以200-240rpm/min在37℃下放置在加热的定轨振荡器上2小时。
    3. 将悬浮液转移到50ml离心管中,然后在190×g离心8分钟。
    4. 弃去上清液,将细胞沉淀重悬于含有15%FBS的新鲜DMEM中。
    5. 然后将细胞接种到已经用5ml聚-D-赖氨酸(10μg/ml)预包被的24孔组织培养板上。将细胞分配到7.5×10 4个细胞/孔中并在37℃下在湿润的5%CO 2中孵育。 span> 气氛。每2-3天用含有10%FBS的新鲜DMEM更换培养基。使用显微镜定期观察细胞生长(图2)。细胞产量为1×10 6个小胶质细胞/小鼠。
    6. 将原始混合胶质细胞烧瓶用10ml含10%FBS的新鲜DMEM补充。此后,使用如上所述的含有10%FBS的新鲜DMEM和烧瓶培养基的上清液的1:1混合物每2-3天更换培养基。然后可以在一周后重复上述纯化过程。在将细胞铺在24孔板中后,细胞必须在一周内使用(对于前3天是最佳的)。可以使用抗CD11b单克隆抗体通过流式细胞术来评估细胞纯度

      图1.从1-3天老鼠移除大脑的方法


      图2.光学显微镜上的小神经胶质

致谢

这项工作是由国家自然科学基金(中国授予30972718和81273242),江苏省自然科学基金(授予BK2012605)和江苏省创新和 创业人才(2011-2014)。

参考文献

  1. Chen,X.,Quinn,E. M.,Ni,H.,Wang,J.,Blankson,S.,Redmond,H. P.,Wang,J.H。和Feng, B7-H3参与实验性肺炎球菌性脑膜炎的发展,通过增加TLR2- 依赖机制。 189(1):347-355。 ,tahoma,verdana,helvetica; font-size:12px; line-height:1.5; text-align:start; text-indent:-17.85pt;">
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, X., Zhang, Y., Sadadcharam, G., Cui, W. and Wang, J. H. (2013). Isolation, Purification, and Culture of Primary Murine Microglia Cells. Bio-protocol 3(1): e314. DOI: 10.21769/BioProtoc.314.
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