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Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during immune reaction. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

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[Bio101] Microglia Cultures and Mixed Glial Culture
[Bio101] 小胶质细胞培养和混合胶质培养

神经科学 > 细胞机理 > 细胞分离和培养
作者: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
11/5/2011, 11460 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.149

[Abstract] Primary rodent microglia-enriched cultures are the most popular model to study microglial biology in vitro and to explore immune signaling pathways. Mixed glial cultures that contain microglia and astroglia are very useful for investigating the precise mechanisms of microglia-astroglia interaction during immune reaction. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

[Abstract] 基本啮齿类小胶质细胞富集培养是体外研究小神经胶质生物学和探索免疫信号途径应用广泛的方法。混合胶质培养宝包括小胶质和星形神经胶质,在研究在免疫反应中小胶质-神经胶质互作的精确机制大有用处。这份指南经过了Dr. Hong实验室不同研究人员尤其是Dr. Bin Liu多年的改进。

Materials and Reagents

  1. Poly-D-lysine (Sigma-Aldrich, catalog number: P7280 )
  2. DMEM/F12 (Life Technologies, InvitrogenTM, catalog number: 11330-032 )
  3. D-Glucose
  4. Sterile water
  5. Sterile PBS
  6. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 16000-044 )
  7. Non-essential amino acids (100 ml) (Life Technologies, Gibco®, catalog number: 11140-050 )
  8. Sodium pyruvate (100 ml) (Sigma-Aldrich, catalog number: S8636 )
  9. 200 mM L-glutamine (100 ml) (Life Technologies, Gibco®, catalog number: 25030-081 )
  10. Penicillin/streptomycin (100 ml) (Sigma-Aldrich, catalog number: P0781 )
  11. DMEM/F12-based culture medium (see Recipes)
  12. Poly-D-lysine stock solution (see Recipes)
  13. Treatment medium (see Recipes)

Equipment

  1. Cell culture incubator
  2. Centrifuges
  3. Dissection microscope
  4. Scissors and forceps
  5. T175 flasks
  6. 70 µm nylon filter (sterile)
  7. 50 ml tube
  8. Sterile filter (0.2 µm)
  9. Foil
  10. Laminar hood

Procedure

  1. Seed cells from 2-2.5 rat brains or 4-5 mouse brains from pups of postnatal day 1 to 5 in to one T175 flask.
  2. Coat T175 flasks with 20 ml Poly-D-lysine (20 µg ml-1) in a laminar hood for 2-3 h or in the in incubator for at least 1 h.
  3. Wash the flasks twice with 25 ml of sterile water. Add 25 ml sterile PBS to each flask.
  4. the animal procedure room, remove whole brains from 1-3 day-old rat or mouse pups and place them in cold DMEM/F12.
  5. Under a microscope, remove olfactory bulb and cerebellum from the brain. Remove meninges and blood vessels.
  6. Pool tissues from up to 10 brains into a 6 cm petri dish and keep dish on ice.
  7. In a laminar hood, transfer tissues to a 50-ml tube. Gently triturate the tissues (5-10 times each) first with 10 ml pipet, then a 1 ml pipet tip fitted to the 10 ml pipet followed with a fitted 200 µl pipet tip.
  8. Filter the minced tissue suspension through a 70 µm nylon filter and centrifuge the filtrate for 10 min at 6.5x speed setting (~1,500 rpm).
  9. Carefully remove the supernatant using 10 ml pipet and resuspend the pelleted cells in 10-20 ml of DMEM/F12-based culture medium.
  10. Completely remove the PBS from flasks. Add 25 ml of pre-warmed (37 °C) DMEM/F12-based culture medium to each flask.
  11. Resuspend the cell suspension again and put the cells to each flask preloaded with medium. Place the flasks into a humidified 37 °C incubator with 5% CO2.
  12. Four days after the initial seeding, remove the medium and add 25 ml of fresh warm culture medium to each flask.
  13. Mixed glia cultures will be ready for shaking off microglia at between 12-14 days after initial seeding.
  14. Separate microglia from astrocytes by shaking the flasks for 5 h at 180 rpm. The enriched microglia are >98% pure as determined by OX-42-immunostaining and glial fibrillary acidic protein (GFAP)-immunostaining.

Recipes

  1. Poly-D-lysine solution
    Dissolve in 50 ml of ddH2O to make 5x stock solution.
    Dilute with sterile ddH2O to 20 µg/ml right before use.
  2. DMEM/F12-based culture medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    430 ml
    -
    Heat-inactivated fetal bovine serum*
    50 ml
    10%
    None essential nonessential amino acids
    5 ml
    0.1 mM
    Sodium pyruvate
    5 ml
    1 mM
    L-glutamine
    5 ml
    2 mM
    Penicillin/streptomycin
    5 ml
    50 U/ml/50 µg/ml
    on.: concentration
    Sterile filter (0.2 µm) and store wrapped in foil at 4 °C.
    *Heat-inactivated at 56 °C for 30 min and stored in 50 ml aliquots at -70 °C.
  3. Treatment medium
    Reagents
    Volume
    Final con.
    DMEM/F12
    470 ml
    -
    Heat-inactivated fetal bovine serum*
    10 ml
    2%
    None essential nonessential amino acids
    5 ml
    0.1 mM
    Sodium pyruvate
    5 ml
    1 mM
    L-glutamine
    5 ml
    2 mM
    Penicillin/streptomycin
    5 ml
    50 U/ml/50 µg/ml
    Sterile filter (0.2 µm) and store wrapped in foil at 4 °C.

References

  1. Gao, H. M., Hong, J. S., Zhang, W. and Liu, B. (2002). Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 22(3): 782-790.
  2. Liu, B., Du, L. and Hong, J. S. (2000). Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. J Pharmacol Exp Ther 293(2): 607-617.

材料和试剂

  1. 聚-D-赖氨酸(Sigma-Aldrich,目录号:P7280)
  2. DMEM/F12(Life Technologies,Invitrogen TM ,目录号:11330-032)
  3. D-葡萄糖
  4. 无菌水
  5. 无菌PBS
  6. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:16000-044)
  7. 非必需氨基酸(100ml)(Life Technologies,Gibco ,目录号:11140-050)
  8. 丙酮酸钠(100ml)(Sigma-Aldrich,目录号:S8636)
  9. 200mM L-谷氨酰胺(100ml)(Life Technologies,Gibco ,目录号:25030-081)
  10. 青霉素/链霉素(100ml)(Sigma-Aldrich,目录号:P0781)
  11. 基于DMEM/F12的培养基(参见配方)
  12. 聚D-赖氨酸储备溶液(见配方)
  13. 处理介质(见配方)

设备

  1. 细胞培养孵化器
  2. 离心机
  3. 解剖显微镜
  4. 剪刀和镊子
  5. T175烧瓶
  6. 70μm尼龙过滤器(无菌)
  7. 50ml管
  8. 无菌过滤器(0.2μm)

  9. 层流罩

程序

  1. 将来自2-2.5大鼠脑的种子细胞或来自出生后第1天的幼仔的4-5只小鼠脑到5个放入一个T175烧瓶中。
  2. 在层流罩中,在具有20mL聚-D-赖氨酸(20μg/ml )的涂层T175烧瓶中孵育2-3小时或在培养箱中至少1小时。
  3. 用25ml无菌水洗涤烧瓶两次。 向每个烧瓶中加入25ml无菌PBS
  4. 动物手术室,从1-3天龄的大鼠或小鼠幼崽移除整个大脑,并将其置于冷DMEM/F12中。
  5. 在显微镜下,从大脑中删除嗅球和小脑。 去除脑膜和血管。
  6. 池组织从多达10个大脑进入一个6厘米的培养皿,并保持在冰上的菜。
  7. 在层流罩,转移组织到50ml管。 轻轻研磨组织(每次5-10次)用10毫升吸管,然后1毫升移液管吸头安装到10毫升吸管,随后用一个适合的200微升移液器吸头。
  8. 通过70μm尼龙过滤器过滤组织悬浮液,并以6.5倍速度(约1,500rpm)离心滤液10分钟。
  9. 使用10ml移液管小心移除上清液,并将沉淀的细胞重悬在10-20ml的DMEM/F12基培养基中。
  10. 完全从烧瓶中取出PBS。 向每个烧瓶中加入25ml预温的(37℃)DMEM/F12基培养基。
  11. 再次悬浮细胞悬浮液,并将细胞放入预先装有培养基的每个烧瓶中。 将烧瓶置于具有5%CO 2的湿润的37℃培养箱中
  12. 初始接种后4天,取出培养基,并向每个烧瓶中加入25ml新鲜的温暖培养基
  13. 混合胶质细胞培养物将准备在初始接种后12-14天之间振荡小胶质细胞。
  14. 通过摇动烧瓶5小时在180 rpm分离小神经胶质细胞从星形胶质细胞。 通过OX-42-免疫染色和胶质纤维酸性蛋白(GFAP) - 免疫染色测定,富集的小胶质细胞纯度> 98%。

食谱

  1. 聚-D-赖氨酸溶液
    溶解在50ml ddH 2 O中以制备5x储备溶液。
    在使用前用无菌ddH 2 O稀释至20μg/ml。
  2. DMEM/F12培养基
    试剂

    最终结果。
    DMEM/F12
    430 ml
    -
    热灭活的胎牛血清*
    50 ml
    10%
    无必需的非必需氨基酸
    5 ml
    0.1 mM
    丙酮酸钠
    5 ml
    1 mM
    L-谷氨酰胺 5 ml
    2 mM
    青霉素/链霉素
    5 ml
    50U/ml /50μg/ml
    on:浓度
    无菌过滤器(0.2μm),存放在4°C的箔中 *在56℃热灭活30分钟,并以50ml等分试样储存于-70℃
  3. 处理介质
    试剂

    最终结果。
    DMEM/F12
    470 ml
    -
    热灭活的胎牛血清*
    10 ml
    2%
    无必需的非必需氨基酸
    5 ml
    0.1 mM
    丙酮酸钠
    5 ml
    1 mM
    L-谷氨酰胺 5 ml
    2 mM
    青霉素/链霉素
    5 ml
    50U/ml /50μg/ml
    无菌过滤器(0.2μm),存放在4°C的箔中

参考文献

  1. Gao,H.M.,Hong,J.S.,Zhang,W.and Liu,B。(2002)。 小胶质细胞在鱼藤酮诱导的多巴胺能神经元变性中的不同作用 Neurosci 22(3):782-790。
  2. Liu,B.,Du,L.and Hong,J.S。(2000)。 纳洛酮通过抑制小胶质细胞活化和超氧化物生成来保护大鼠多巴胺能神经元免受炎症损伤。 J Pharmacol Exp Ther 293(2):607-617。
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How to cite this protocol: Gao, H. (2011). Microglia Cultures and Mixed Glial Culture. Bio-protocol Bio101: e149. DOI: 10.21769/BioProtoc.149; Full Text



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3/10/2014 6:31:30 PM  

Yoojin Seo
Seoul national univ.

Hi, thank you for your kind instruction. I followed yours but i cannot get enough microglial cells... How many cells can you usually get using shaking methods? I seeded whole cells from 1 neonate mouse onto 75 T flask and maintain culture for 14 days....

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4/6/2012 12:05:55 PM  

Is it M-CSF or GM-CSF necessary for the microglia culture?

4/7/2012 1:43:51 PM  

Huiming Gao (Author)
Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA

M-CSF and GM-CSF have been found to induce microglial proliferation in fetal and adult cultures. However, we have never added exogenous M-CSF or GM-CSF into our culture medium. It is possible that low levels of M-CSF and/or GM-CSF in fetal bovine serum may help microglia cultures grow.

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8/15/2011 3:38:37 PM  

Yuanqing Lin
Test institute

explan i n brief plz,......
Coat T175 flasks with 20 ml Poly-D-lysine (20 µg/ml) in a laminar hood for 2-3 hr or in the in incubator for at least 1 hour.

regards

robin.hood008@yahoo.com

8/31/2011 4:04:51 PM  

bio-protocol

For the sterile purpose, in a laminar hood, add 20 ml of 20 µg/ml Poly-D-lysine into T175 flasks. leave the flasks in the laminar hood for 2-3 hrs or put the flasks into incubator for 1 hour. then remove the Poly-D-lysine from flasks and wash the flasks as described.

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