大鼠胚胎神经细胞的分离和培养：一个快速议定书“
Summary
我们描述了一个快速的方法分离和培养的海马和皮层神经细胞从老鼠胚胎。该协议允许在几乎纯神经细胞需要我们进行实验。
Abstract
我们描述了一个快速的方法分离和培养E15-17大鼠胚胎海马或皮层神经元。程序可以成功地应用于老鼠和人类的初级神经元和神经祖细胞的分离。分离神经元无血清培养基均保持在几个星期。这些文化可用于核转染，免疫细胞化学，核酸制备，以及电。年老的神经细胞，也可以慢病毒转导效率，有效地减少，磷酸钙或脂质的方法，如脂质体转染。
Protocol
1。聚-D-赖氨酸（PDL）的制备加入5毫升无菌DDH 2至5毫克的客运专线，获得1毫克/毫升的原液。 混合原液吹打几次。 立即使用或储存聚-D-赖氨酸的解决方案，在2-8°C。 2。聚-D-赖氨酸（PDL）：涂层塑料细胞培养皿 PDL原液稀释用无菌DDH 2 O的终浓度为10微克/毫升。 吸取足够的解决方案，覆盖养殖水面面积为60毫米的菜（3毫?…
Discussion
大鼠海马和皮层神经元的解剖和文化在这里介绍的方法，允许使用在化学上定义的介质（ 图3）增长了近纯神经细胞进行实验。虽然近纯无血清培养基中培养神经元的协议已先前所述的2,3,4，也有我们的方法中作出了重要的变化。不同于传统的协议（即银行家等）5，我们已取代TrypLE快速，更温和的分解酶胰蛋白酶。我们也省略了两个步骤，这可能会影响神经细胞的完?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢社论援助乔娜埃利斯。项目描述奖号码R01MH079751（主持人：F.佩鲁齐）从国家心理健康研究所的支持。内容完全是作者的责任，并不代表国家精神卫生研究所或国立卫生研究院的官方意见。
Materials
| Reagent | Concentration |
| Neurobasal | 98% |
| B27 | 2% |
| Glutamax | 0.5 mM |
Table I. Neurobasal/B27 complete medium.
| Reagent | Concentration |
| Glucose | 16 mM |
| Sucrose | 22 mM |
| HEPES | 10 mM |
| NaCl | 160 mM |
| KCl | 5 mM |
| Na2HPO4 | 1 mM |
| KH2PO2 | 0.22 mM |
| Gentamicin | 50 μg/ml |
| Fungizone | 250 ng/ml |
| pH | 7.4 |
| Osmolarity | 320-330 mOsm |
Table II. Dissection medium.
| Reagent | Volume (μl) |
| Neurobasal/B27 complete medium | 240 |
| Trypan Blue Stain 0.4% | 250 |
| Total | 490 |
Table III. 50x Counting solution.
| Reagent | Company | Cat. number |
| Hibernate E | Brainbits | 767171 |
| Neurobasal | Gibco, Invitrogen | 21103-049 |
| B27 | Gibco, Invitrogen | 17504-044 |
| Fungizone | Gibco, Invitrogen | 15290-018 |
| Gentamicin sulfate | Sigma Aldrich | G1264 |
| Glutamax 200 mM | Gibco, Invitrogen | 35050 |
| TrypLE Express w/o phenol red | Gibco, Invitrogen | 12604 |
| Cytosine-β-D-arabinofuranoside hydrochloride | Sigma Aldrich | C6645 |
| Poly-D-Lysine | Sigma Aldrich | P6407 |
| Laminin 1 mg/ml | Millipore | CC095 |
| HEPES | Sigma Aldrich | H3375 |
| Trypan Blue Stain 0.4% | Gibco, Invitrogen | 15250 |
Table IV. Specific reagents.
| Equipment | Company | Cat. number |
| Stereo Microscope | Olympus | SZ61 |
| Large Forceps | FST | 11022-14 |
| Fine-tipped forceps | Moria | MC40B |
| Micro fine-tipped forceps | Moria | MC31 |
| Razor-sharp scissors | Roboz | RS-6820 |
| Micro Dissecting scissors | FST | 91460-11 |
| Micro Dissecting Curved scissors | FST | 14067-11 |
| Glass 2-chamber slides | Lab-Tek | 154461 |
| 60 mm dishes | BD Falcon | 353002 |
| 100 mm dishes | Corning | 430167 |
| 15 ml tubes | BD Falcon | 352099 |
| 1.5 ml cryo-tube vial | Nunc | 375353 |
Table V. Specific equipment.
References
- Aprea, S. Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage. J. Neurosci. 26, 4054-4062 (2006).
- Kivell, B. M., McDonald, F. J., Miller, J. H. Serum-free culture of rat post-natal and fetal brainstem neurons. Brain Res. Dev. Brain Res. 120, 199-210 (2000).
- Kivell, B. M., McDonald, F. J., Miller, J. H. Method for serum-free culture of late fetal and early postnatal rat brainstem neurons. Brain Res. Brain Res. Protoc. 6, 91-99 (2001).
- Brewer, G. J. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674-683 (1995).
- Banker, G., Goslin, K. . Culturing nerve cells. , (1998).
- Eletto, D. Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a. J. Cell Physiol. 216, 764-770 (2008).
- Gualco, E. IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors. Cell Death Differ. 17, 439-451 (2010).
- Gage, F. H. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).
- Keyser, D. O., Pellmar, T. C. Synaptic transmission in the hippocampus: critical role for glial cells. Glia. 10, 237-243 (1994).
- Pfrieger, F. W., Barres, B. A. Synaptic efficacy enhanced by glial cells in vitro. Science. 277, 1684-1687 (1997).
Tags
IsolationCultureRat Embryonic Neural CellsQuick ProtocolDissociationHippocampal NeuronsCortical NeuronsE15-17 Rat EmbryosMouse Primary NeuronsHuman Primary NeuronsNeural ProgenitorsSerum-free MediumNucleofectionImmunocytochemistryNucleic Acids PreparationElectrophysiologyLentiviral TransductionCalcium Phosphate TransfectionLipid-based Transfection
Cite This Article
Pacifici, M., Peruzzi, F. Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol. J. Vis. Exp. (63), e3965, doi:10.3791/3965 (2012).