大鼠皮质原代神经元和胶质细胞共培养作为中枢神经系统神经炎症的研究模型

Ya-Ni Huang; Jia-Yi Wang

doi:10.21769/BioProtoc.1788

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Peer-reviewed

Primary Neuron-glia Culture from Rat Cortex as a Model to Study Neuroinflammation in CNS Injuries or Diseases

大鼠皮质原代神经元和胶质细胞共培养作为中枢神经系统神经炎症的研究模型

Ya-Ni Huang

Jia-Yi Wang email

发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1788 浏览次数: 11162

评审: Soyun KimPengpeng LiHong-guang Xia

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Cover of Journal of Neuroinflammation, featuring study using the protocol.

Aug 2015

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Abstract

Primary neuron-glia cultures are commonly used in vitro model for neurobiological studies. Here, we provide a protocol for the isolation and culture of neuron-glial cells from cortical tissues of 1-day-old neonatal Sprague-Dawley pups. The procedure makes available an easier way to obtain the neuron and glia. In this culture system, neuron-glia cultures consisted of approximately 37% neurons, 51% astrocytes, 7% microglia, and a small percentage (<5%) of other cells after fourteen days in vitro. Primary neuron-glia cultures is a simplified in vitro model for studies focusing on interactions between neurons and glia cells. Activated glial cells, mainly astrocytes and microglia, are histopathological hallmarks of acute injury of the central nervous system (CNS) or chronic neurologic diseases (Hirsch and Hunot, 2009; Lee et al., 2009; Minghetti, 2005). Inflammatory mediators (e.g., nitric oxide, reactive oxygen species, proinflammatory cytokines, and chemokines) released by activated glia can directly or indirectly cause neuronal damage or neurodegeneration. Neuroinflammation is a common mechanism of various neurological diseases leading to neurodegeneration. The advantages of neuron-glia cultures are that: (1) Cultured cells can bypass complicated physiological interactions (such as leukocyte infiltration, blood-brain barrier, reflex or other systemic regulation) in vivo to allow direct observation of neuroinflammation caused by various CNS insults (hypoxia, ischemia, trauma. infection, neurotoxins, chronic stress or diseases); (2) Unlike cell lines that are mostly derived from tumor cells, primary cultured neuron-glia system is closer to the cell population ratio in vivo and can mimic the in situ microenvironment; and (3) Cultures can be prepared from various brain regions (e.g., cortex, hippocampus, mesencephalon…etc.) and allow an opportunity to examine the regional difference in the susceptibility to neurodegeneration following neuroinflammation caused by various CNS insults (Kim et al., 2000). The following protocol is an example for primary rat cortical neuron-glia culture preparation (Huang et al., 2015; Huang et al., 2014; Huang et al., 2012; Huang et al., 2009).

Keywords: Primary neuron-glia culture (原发性神经胶质细胞培养)

Neuroinflammation (神经炎症)

CNS injuries (中枢神经系统损伤)

I (我)

Materials and Reagents

Tissue culture dishes (60 x 15 mm) (Sigma-Aldrich, catalog number: P5237 )
Tissue culture dishes (100 x 20 mm) (Nunc, catalog number: 172958 )
Screw cap centrifuge tube (50 ml) (Sigma-Aldrich, catalog number: BR114821 )
24-well plates (Sigma-Aldrich, catalog number: CLS 3527 )
Pipette tips (10 μl, 200 μl and 1,000 μl) (Shineteh instruments co ltd., catalog number: PT4-W10 , PT1-Y200 and PT5-W10 )
Neonatal Sprague-Dawley pups (1-day-old)
Trypan blue (Thermo Fisher Scientific, Gibco^TM, catalog number: 15250061 )
Hanks’ Balanced Salt solution (HBSS) (Sigma-Aldrich, catalog number: 55021C )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
Pyruvate (Sigma-Aldrich, catalog number: P2256 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Bovine Serum Albumins (BSA) (Sigma-Aldrich, catalog number: A9418 )
DMED powder (Thermo Fisher Scientific, Gibco^TM, catalog number: 12100-046 )
DMED/F-12, HEPES, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 11039-021 )
Penicilline/Stretomycin (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
100 mM Sodium pyruvate solution (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360-070 )
MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140-050 )
Fetal bovine serum (FBS) (NQBB, catalog number: A6806-11 )
Hank’s solution (see Recipes)
Dulbecco's modified Eagle’s medium (DMEM) (see Recipes)
Serum-free medium (100 ml) (see Recipes)

Equipment

37 °C, 5% CO₂ incubator (Water Jacketed Laboratory CO₂ Incubator)
Stereo microscope (Shineteh instruments co., catalog number: IH1-ZM150A )
Biological Inverted microscope (OLYMPUS CORPORATION, model: IX71 )
Mini micro centrifuge (Shineteh instruments co., catalog number: IC-MINIMAX )
Centrifuge (Hermle, catalog number: Hermle Z232K )
Water bath (Bioman Scientific Co Ltd, catalog number: SWB-20-1 )
Counting chamber (Shineteh instruments co., catalog number: PT14-901001 )
Operating scissors STR (Shineteh instruments co., catalog number: ST-014 )
Iris scissors STR (Shineteh instruments co., catalog number: ST-S009 )
Dressing forceps (Shineteh instruments co., catalog number: ST-D114 )
Iris forceps (Shineteh instruments co., catalog number: ST-I510 )
Tweezers forceps (Shineteh instruments co ltd., catalog number: ST-NO5 )
Pipetman (Gilson, catalog number: P10 , P200 and P1000)
Pipet-aid (Thomas Scientific, Flacon^TM, catalog number: 0410C04 )
Pipets (10 ml) (Tseng Hsiang Life Science ltd., catalog number: SP-1-C )
Ice bucket (Shineteh instruments co ltd., catalog number: PA8-4 )

Procedure

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如何引用

Huang, Y. and Wang, J. (2016). Primary Neuron-glia Culture from Rat Cortex as a Model to Study Neuroinflammation in CNS Injuries or Diseases. Bio-protocol 6(8): e1788. DOI: 10.21769/BioProtoc.1788.