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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, 10306 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.

材料与试剂

 

1.        -D-赖氨酸 (Sigma P-7280)

2.        DMEM/F12 (Invitrogen 11330-032)

3.        D-葡萄糖

4.        热灭活胎牛血清(FBS; Invitrogen/Gibco 16000-044)

5.        不含非必要氨基酸 (Invitrogen /Gibco 11140-050; 100 ml)

6.        丙酮酸钠 (Sigma; S8636; 100 ml)

7.        L-谷氨酰胺 (Invitrogen /Gibco 25030-081; 100ml; 200 mM)

8.        青霉素/链霉素 (Sigma P0781; 100 ml)

 

仪器

 

1.          细胞培养孵化器

2.          离心机

3.          解剖镜

4.          见到和镊子

5.          T175烧瓶

6.          70 μm 尼龙过滤器(灭菌)

7.          无菌过滤器 (0.2 μm)

8.          铝箔

 

步骤

 

1.          2-2.5天的大鼠大脑细胞或出4-5出生后第1-5的小鼠幼仔大脑置于T175烧瓶中培养

2.          T175烧瓶里涂布20 ml- D -赖氨酸(20 μg/ml) 层流罩上晾2-3在孵化器至少1小时。

3.          两次用25 ml无菌水洗涤烧瓶。每个烧瓶中加入25 ml无菌PBS

4.          在动物实验室中,从1-3日龄大鼠或小鼠的幼崽中除整个大脑并放在预冷的DMEM/F12

5.          在显微镜下,去除脑中的嗅球和小脑。除去脑膜和血管。将从近10大脑中取得的组织置于一个6cm培养皿中,置于冰上。

6.          在层流罩,将组织转移到一个50ml。轻轻磨碎组织(每5-10。先用10ml的移液管,然后依次用和其配套的1ml200 μl枪头。

7.          70μm尼龙过滤器过滤组织悬液, 6.5X转速1500 RPM离心滤液

8.          小心用10ml移液管管取出上清液,并10-20 mlDMEM/F12培养基重新悬浮细胞沉淀

9.          彻底PBS从烧瓶中去除。添加25ml预热37DMEM/F12培养基到每个烧瓶中。

10.      再次重悬细胞,并把细胞装入预先倒入培养基的每个烧瓶。烧瓶放入5CO2 37保湿培养箱。

11.      开始培养4天,去除培养基,添加25ml的新鲜且预热的培养基每个烧瓶。

12.      初始培养12-14天之间摇除小胶质细胞混合胶质细胞培养就算完成 

通过180 rpm摇动烧瓶5hrs可以将小胶质细胞星形胶质细胞分离。富的小胶质细胞将达到>98%的纯度。这可通过OX- 42和神经胶质纤维酸性蛋白(GFAP)的免疫染色测定出

 

配方

 

1.      D-赖氨酸 (Sigma P-7280)50 mlddH2O溶解,配成 5X母液。使用前用灭菌ddH2O稀释 20 μg/ml

2.        DMEM/F12培养基

试剂                                                                             体积                 终浓度

DMEM/F12                                                                   430 ml              -

热灭活胎牛血清*                                                            50 ml                10%

不含非必须氨基酸                                                          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 (0.2 μm)并用铝箔包裹存于 4°C

* 56°C热灭活 30 min并分装成 50ml保存于 –70°C

3.        处理培养基

试剂                                                                             体积                终浓度

DMEM/F12                                                                   470 ml              -

热灭活胎牛血清*                                                            10 ml                2%

不含非必须氨基酸                                                          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

0.2 μm滤器过滤除菌并用铝箔包裹保存于 4°C

 

参考文献

 

1.         Liu B., Du L., Hong J.S. (2000). Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation. Journal of Pharmacology and Experimental Therapeutics 293(2): 607-17. 

2.         Gao H.M., Hong J.S., Zhang W., Liu B. (2002). Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. Journal of Neuroscience 22(3): 782-90.  

 

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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  

Anonymous 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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