Isolation and Maintenance of Murine Embryonic Striatal Neurons
小鼠胚胎纹状体神经元的分离与维持
发布: 2018年04月20日第8卷第8期 DOI: 10.21769/BioProtoc.2823 浏览次数: 11101
评审: Oneil G. BhalalaTatiana Rosado RosenstockRyohei Iwata
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The authors used this protocol in:
Sep 2017
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Abstract
Primary cultures of murine striatal neurons are widely used to explore cellular mechanisms in neurobiology, including brain diseases. Here we describe a detailed and standardized protocol to dissect and culture embryonic murine striatal neurons GABA-positive/DARPP-32-positive for 12 days in vitro, when they show good neuronal cell connectivity and the presence of dendritic spines, which reflects the maturation of the network.
Keywords: Primary cultures (原代培养物)
Striatum dissection (纹状体解剖)
Striatal neurons (纹状体神经元)
Medium spiny neurons (中型多棘神经元)
Background
Striatum is a critical component of the motor and reward systems and dysfunction of striatal neurons can lead to a variety of neuronal disorders, from obsessive-compulsive-like behaviors (Welch et al., 2007) to neurodegeneration, as observed in Huntington’s disease (Reiner et al., 1988). Therefore, a well-stablished striatal culture may be a great value as a model for studying these and other conditions. The major neuronal subtype in adult striatum is medium spiny projection neurons (MSNs), which constitutes approximately 95% of all striatal neurons and use the inhibitory transmitter gamma-aminobutyric acid (GABA). They are characterized by medium-sized cell bodies, complex dendritic arbors and a high density of dendritic spines that receive both glutamatergic and dopaminergic inputs. The remaining 5% of neurons are composed by GABAergic aspiny interneurons (Graveland and DiFiglia, 1985; Matamales et al., 2009). The following protocol describes the preparation of primary cultures of mouse embryonic striatal neurons with a low percentage of astrocytes, which proliferation is prevented by the mitosis inhibitor 5-fluoro-2’-deoxyuridine (5-FdU). After 12 days in vitro (DIV) the striatal neurons obtained by the described protocol are expected to be immunolabeled for microtubule-associated protein 2 (MAP2) or NeuN, GABA and a large percentage for dopamine- and cAMP-regulated phosphoprotein (DARPP-32).
Materials and Reagents
- Coverslips 18 mm diameter round, #1.5 (0.17 mm) thickness (Thermo Fisher Scientific, Menzel, catalog number: 11817742 )
- Sterile non-tissue culture treated Petri dishes of 35, 60 and 100 mm (VWR, catalog numbers: 734-1707 , 734-1708 , 734-1709 )
- 5, 10 ml serological sterile pipettes (Labbox, catalog numbers: MPIP-U05-200 , MPIP-U10-200 )
- Sterile 0.2-10 μl, 10-200 μl and 100-1,000 μl micropipette tips (Frilabo, catalog numbers: 170558 , 171017 , 171024 )
- 15 and 50 ml sterile conical centrifuge tubes (Corning, catalog numbers: 430791 , 430829 )
- Sterile 1.5 ml microcentrifuge tubes (BIOplastics, catalog number: B74085 )
- Sterile syringes, 20 ml (Terumo, catalog number: SS+20L1 )
- Sterile 230 mm glass Pasteur pipettes (Labbox, catalog number: PIPN-230-250 )
- Sterile 0.2 μm acetate cellulose filters (GE Healthcare, Whatman, catalog number: 28415732 )
- Vacuum 0.2 μm filter bottle system, 500 ml (Corning, catalog number: 430769 )
- Cell strainers, 40 μm (Corning, Falcon®, catalog number: 352340 )
- Multi-wells tissue culture treated, flat bottom, polystyrene (Corning, catalog numbers: 3596 , 3548 , 3524 , 3512 , 3506 )
- Pregnant female mice (Mus musculus) with 16 days of gestation
Note: In this protocol, mice from FVB/NJ inbred strain (THE JACKSON LABORATORY) were used. - Hydrochloric acid (HCl), 37% (Sigma-Aldrich, catalog number: 435570 )
- Ethanol absolute 99.8% (Fisher Scientific, catalog number: 10342652 )
- Isoflurane Iso-Vet 1,000 mg/g Inhalation Vapour (Chanelle UK)
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
- Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
- 5-FdU (Sigma-Aldrich, catalog number: F0503 )
- Anti-MAP2 (1:500; Santa Cruz Biotechnology, catalog number: sc-32791 )
- Anti-GABA (1:500; Sigma-Aldrich, catalog number: A2052 )
- Anti-DARPP32 (1:100; Abcam, catalog number: ab40801 )
- Goat anti-rabbit IgG secondary antibody, Alexa Fluor 594 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37117 )
- Donkey anti-mouse IgG secondary antibody, Alexa Fluor 488 (1:200; Thermo Fisher Scientific, Invitrogen, catalog number: R37114 )
- Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
- Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P1149 )
- Potassium chloride (KCl) (Labbox, catalog number: POCL-00P-1K0 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
- Sodium chloride (NaCl) (Merck, catalog number: 106404 )
- Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S5761 )
- Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
- D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270 )
- Sodium pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
- HEPES (Sigma-Aldrich, catalog number: H3375 )
- Phenol red (Sigma-Aldrich, catalog number: P3532 )
- Deoxyribonuclease I (DNase I) (Sigma-Aldrich, catalog number: D5025 )
- Bovine serum albumin (BSA) fatty acid free (Sigma-Aldrich, catalog number: A6003 )
- Trypsin, Type IV-S, from porcine pancreas (Sigma-Aldrich, catalog number: T0303 )
- Trypsin inhibitor, type II-S: Soybean (Sigma-Aldrich, catalog number: T9128 )
- L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
- Gentamicin (50 mg/ml) (Thermo Fisher Scientific, catalog number: 15750060 )
- Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
- B-27 supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
- Poly-D-lysine for coating (0.1 mg/ml) (see Recipes)
- Hanks’ balanced salt solution (HBSS) (see Recipes)
- DNase I solution (5 mg/ml) (see Recipes)
- BSA solution (3 mg/ml) (see Recipes)
- Trypsin solution (0.5 mg/ml) (see Recipes)
- Trypsin inhibitor solution (1 mg/ml) (see Recipes)
- Washing solution (see Recipes)
- Inactivated FBS (see Recipes)
- Supplemented Neurobasal medium (see Recipes)
Equipment
- 11.5 cm fine straight scissor (Fine Science Tools, Dumont, catalog number: 14060-11 )
- 8.5 cm mini-dissecting scissor, straight with sharp tip (World Precision Instruments, catalog number: 503669 )
- Medium forceps (Fine Science Tools, model: Dumont #7, catalog number: 11274-20 )
- Forceps with straight and angled fine tip (Fine Science Tools, models: Dumont #5 and Dumont #5/45 )
- Automatic pipettor and micropipettes
- Glass Schott laboratory bottle with cap, 500 ml (DWK Life Sciences, Duran, catalog number: 21 801 44 5 )
- Water bath with electronic temperature regulation
- Ultrasonic cleaning bath (just for coverslips washing)
- Zeiss Stemi magnification glass (or equivalent)
- Phase contrast inverted microscope equipped with 10x and 20x objectives
- Zeiss LSM 710 point-scanning confocal microscope (ZEISS, model: LSM 710 or equivalent)
- Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
- Water jacketed CO2 incubator
- Vertical laminar flow chamber
- Isoflurane vaporizer apparatus (E-Z Anesthesia, model: EZ-SA800 , or equivalent)
Software
- Fiji software (ImageJ, National Institute of Health, USA)
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Naia, L. and Rego, A. C. (2018). Isolation and Maintenance of Murine Embryonic Striatal Neurons. Bio-protocol 8(8): e2823. DOI: 10.21769/BioProtoc.2823.
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分类
神经科学 > 细胞机理 > 细胞分离和培养
细胞生物学 > 细胞分离和培养
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