小鼠脊髓背角神经元的原代培养

De-Li Cao; Peng-Bo Jing; Bao-Chun Jiang; Yong-Jing Gao

doi:10.21769/BioProtoc.2098

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Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn

小鼠脊髓背角神经元的原代培养

DC De-Li Cao

PJ Peng-Bo Jing

BJ Bao-Chun Jiang

YG Yong-Jing Gao email

发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2098 浏览次数: 16461

评审: Jia LiGeoff LauFabiana Scornik

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Cover of The Journal of Clinical Investigation, featuring study using the protocol.

Feb 2016

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Abstract

Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.

Keywords: Neuron (神经元)

Background

Unlike existing protocols for culturing isolated mice primary neurons from hippocampus or cerebral cortex, few methods of culturing SDHN in vitro have been reported. This protocol was mainly based on previously described methods (Hu et al., 2003; Hugel and Schlichter, 2000). Here we made a few modifications including reagents, recipes, dissection and described step-by-step procedures of the dissection and culture of primary SDHN from newborn mice. In this protocol, neurons were gained using the enzymatic (papain) digestion method from fresh spinal dorsal horn tissues directly. The culture of SDHN in vitro can be used for further experiments, such as electrophysiological recordings, immunocytochemistry, and Ca²⁺ imaging, which better support cell behaviors in the spinal cord.

Materials and Reagents

Coverslips (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 174950 )
24-well cell culture plate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 142475 )
Cell culture dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 153066 )
Sterile centrifuge tubes (5 ml and 50 ml)
Cotton ball
Pipette tip
Cell strainer (40 μm) (Corning, Falcon^®, catalog number: 352340 )
Ice
3 days-old mice
Diethyl ether (Sigma-Aldrich, catalog number: 346136 )
75% ethanol
Cytosine arabinoside (Sigma-Aldrich, catalog number: C6645 )
4% formaldehyde
5% goat serum
MAP2 antibody (Sigma-Aldrich, catalog number: M1406 )
Anti-mouse FITC-conjugated secondary antibody
DAPI (Sigma-Aldrich, catalog number: M9542 )
Note: This product has been discontinued.
Collagen (Sigma-Aldrich, catalog number: C7661 )
Poly-D-lysine (Sigma-Aldrich, catalog number: P7405 )
HEPES (1 M) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630080 )
HBSS (Thermo Fisher Scientific, Gibco^TM, catalog number: 14025092 )
Papain (Worthington Biochemical, catalog number: LS003119 )
Neuro basal (Thermo Fisher Scientific, Gibco^TM, catalog number: 10888022 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 1099141 )
Heat-inactivated horse serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 26050070 )
B27 (50x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504044 )
Glutamax (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )
Penicillin/Streptomycin (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Collagen stock solution (see Recipes)
Poly-D-lysine (PDL) stock solution (see Recipes)
Coating solution (see Recipes)
HBSS + HEPES solution (see Recipes)
Papain solution (see Recipes)
Culture media (see Recipes)

Equipment

Surgical scissors (decapitation/dissecting spinal column from body) (RWD Life Science, catalog number: S14014-14 )
Blunt-tipped forceps (dissecting/holding spinal column) (RWD Life Science, catalog number: F13017-12 )
Long and narrow-tipped spring scissors (dissecting spinal cord/cutting nerve roots) (66 Vision, catalog number: 54053B )
Small-tipped spring scissors (cutting dura) (Fine Science Tools, catalog number: 15000-02 )
Microforceps (holding spinal cord/trimming off dura) (World Precision Instruments, catalog number: 14098 )
Scalpel #15 sterilized surgical blade (cutting spinal cord to isolate dorsal horn) (Shanghai Jinhuan Medical, catalog number: YY0174-2005 )
Tissue culture hood (Suzhou Antai Airtech, model: SW-CJ-1F(D) )
Stereomicroscope (OLYMPUS, model: SZ61 )
Benchtop centrifuge (4 °C, 50 ml tube) (Eppendorf, model: 5430R )
Pipette (Eppendorf, model: ES-1000 )
Haemocytometer (Shanghai anxin optical instrument manufacture, model: XB-K-25 )
Stackable CO₂ incubator (Eppendorf, model: Galaxy^®170R )

Procedure

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Cao, D., Jing, P., Jiang, B. and Gao, Y. (2017). Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn. Bio-protocol 7(1): e2098. DOI: 10.21769/BioProtoc.2098.