Name: an enhanced green fluorescence protein-based assay for studying neurite outgrowth in primary neurons
Uploaded: 2019-10-19T13:00:00
Description: Watch this Scientific Journal Video about An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons at JoVE.com

这项工作由香港研究资助局、健康及医学研究基金（香港）、中大直接资助计划、联合学院捐赠基金及TUYF慈善信托基金资助。

所遵循的所有程序均符合香港中文大学动物实验伦理委员会的道德标准。
1. 准备盖玻片
<ol><li>将无菌的 18 mm 圆形盖玻片放入 12 孔组织培养板的每个孔中。</li>
	<li>在加湿的 37°C 培养箱中用 5 μg/mL 多 D-莱金溶液涂抹盖玻片，至少 1 小时。</li>
	<li>从组织培养板中吸出多D-莱辛溶液，用无菌水冲洗一次涂层盖玻片。</li>
</ol>2. 大鼠胚胎神经元解剖
<ol><li>在妊?...

<ol>
	<li>Kaplan, A., Bueno, M., Hua, L., Fournier, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+functional+axon+repair+in+the+injured+central+nervous+system:+Lessons+from+neuronal+development.">Maximizing functional axon repair in the injured central nervous system: Lessons from neuronal development.</a> Developmental Dynamics. 247 (1), 18-23 (2018).</li><li>Harrill, J. A., Mundy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+neurite+outgrowth+in+PC12+cells.">Quantitative assessment of neurite outgrowth in PC12 cells.</a> Methods in Molecular Biology. 758, 331-348 (2011).</li><li>Yeyeodu, S. T., Witherspoon, S. M., Gilyazova, N., Ibeanu, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+inexpensive+high+throughput+screen+method+for+neurite+outgrowth.">A rapid, inexpensive high throughput screen method for neurite outgrowth.</a> Current Chemical Genomics. 4, 74-83 (2010).</li><li>Ben-David, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+transcriptional+evolution+alters+cancer+cell+line+drug+response.">Genetic and transcriptional evolution alters cancer cell line drug response.</a> Nature. 560 (7718), 325-330 (2018).</li><li>Edwards, M. A., Loxley, R. A., Williams, A. J., Connor, M., Phillips, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+functional+expression+of+NMDA+receptors+in+PC12+cells.">Lack of functional expression of NMDA receptors in PC12 cells.</a> Neurotoxicology. 28 (4), 876-885 (2007).</li><li>LePage, K. T., Dickey, R. W., Gerwick, W. H., Jester, E. L., Murray, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+neuro-2a+neuroblastoma+cells+versus+intact+neurons+in+primary+culture+for+neurotoxicity+studies.">On the use of neuro-2a neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies.</a> Critical Reviews in Neurobiology. 17 (1), 27-50 (2005).</li><li>Karra, D., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+for+neuronal+cells.">Transfection techniques for neuronal cells.</a> Journal of Neuroscience. 30 (18), 6171-6177 (2010).</li><li>Cheung, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FE65+interacts+with+ADP-ribosylation+factor+6+to+promote+neurite+outgrowth.">FE65 interacts with ADP-ribosylation factor 6 to promote neurite outgrowth.</a> The FASEB Journal. 28 (1), 337-349 (2014).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+adaptor+FE65+stimulates+Rac1-mediated+neurite+outgrowth+by+recruiting+and+activating+ELMO1.">Neuronal adaptor FE65 stimulates Rac1-mediated neurite outgrowth by recruiting and activating ELMO1.</a> Journal of Biological Chemistry. 293 (20), 7674-7688 (2018).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Meijering, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+tool+for+neurite+tracing+and+analysis+in+fluorescence+microscopy+images.">Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.</a> Cytometry. Part A: the Journal of the International Society for Analytical Cytology. 58 (2), 167-176 (2004).</li><li>Swanson, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+maps+4.0-Structure+of+the+rat+brain:+An+open+access+atlas+with+global+nervous+system+nomenclature+ontology+and+flatmaps.">Brain maps 4.0-Structure of the rat brain: An open access atlas with global nervous system nomenclature ontology and flatmaps.</a> The Journal of Comparative Neurology. 526 (6), 935-943 (2018).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> Journal of Neuroscience Research. 42 (5), 674-683 (1995).</li><li>Yamada, K. M., Spooner, B. S., Wessells, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+growth:+roles+of+microfilaments+and+microtubules.">Axon growth: roles of microfilaments and microtubules.</a> Proceedings of the National Academy of Sciences of the United States of America. 66 (4), 1206-1212 (1970).</li><li>Casella, J. F., Flanagan, M. D., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochalasin+D+inhibits+actin+polymerization+and+induces+depolymerization+of+actin+filaments+formed+during+platelet+shape+change.">Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change.</a> Nature. 293 (5830), 302-305 (1981).</li><li>Calabrese, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+and+regulating+neurite+outgrowth.">Enhancing and regulating neurite outgrowth.</a> Critical Reviews in Toxicology. 38 (4), 391-418 (2008).</li><li>Lau, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexras1+Interacts+with+FE65+to+Regulate+FE65-Amyloid+Precursor+Protein-dependent+Transcription.">Dexras1 Interacts with FE65 to Regulate FE65-Amyloid Precursor Protein-dependent Transcription.</a> Journal of Biological Chemistry. 283 (50), 34728-34737 (2008).</li><li>Cui, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niacin+treatment+of+stroke+increases+synaptic+plasticity+and+axon+growth+in+rats.">Niacin treatment of stroke increases synaptic plasticity and axon growth in rats.</a> Stroke. 41 (9), 2044-2049 (2010).</li><li>Khodosevich, K., Monyer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+involved+in+neurite+outgrowth+of+postnatally+born+subventricular+zone+neurons+in+vitro.">Signaling involved in neurite outgrowth of postnatally born subventricular zone neurons in vitro.</a> BMC Neuroscience. 11, 18 (2010).</li><li>Tang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resveratrol+Enhances+Neurite+Outgrowth+and+Synaptogenesis+Via+Sonic+Hedgehog+Signaling+Following+Oxygen-Glucose+Deprivation/Reoxygenation+Injury.">Resveratrol Enhances Neurite Outgrowth and Synaptogenesis Via Sonic Hedgehog Signaling Following Oxygen-Glucose Deprivation/Reoxygenation Injury.</a> Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 43 (2), 852-869 (2017).</li><li>He, W., Liu, Y., Tian, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosuvastatin+Improves+Neurite+Outgrowth+of+Cortical+Neurons+against+Oxygen-Glucose+Deprivation+via+Notch1-mediated+Mitochondrial+Biogenesis+and+Functional+Improvement.">Rosuvastatin Improves Neurite Outgrowth of Cortical Neurons against Oxygen-Glucose Deprivation via Notch1-mediated Mitochondrial Biogenesis and Functional Improvement.</a> Frontiers in Cellular Neuroscience. 12, 6 (2018).</li><li>Tesarova, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+for+advanced+glycation+end+products+(RAGE)--soluble+form+(sRAGE)+and+gene+polymorphisms+in+patients+with+breast+cancer.">Receptor for advanced glycation end products (RAGE)--soluble form (sRAGE) and gene polymorphisms in patients with breast cancer.</a> Cancer Investigation. 25 (8), 720-725 (2007).</li><li>Park, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocalcin+Promotes+Neuronal+Differentiation+and+Inhibits+Astrocytic+Differentiation+in+Neural+Stem+Cells.">Hippocalcin Promotes Neuronal Differentiation and Inhibits Astrocytic Differentiation in Neural Stem Cells.</a> Stem Cell Reports. 8 (1), 95-111 (2017).</li><li>Radbruch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence:+Basic+Considerations.">Immunofluorescence: Basic Considerations.</a> Flow Cytometry and Cell Sorting. , 38-52 (2000).</li><li>Wang, T., Larcher, L. M., Ma, L., Veedu, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Screening+of+Commonly+Used+Commercial+Transfection+Reagents+towards+Efficient+Transfection+of+Single-Stranded+Oligonucleotides.">Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides.</a> Molecules. 23 (10), (2018).</li><li>Sariyer, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+neuronal+cultures.">Transfection of neuronal cultures.</a> Methods in Molecular Biology. 1078, 133-139 (2013).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Research. 126 (3), 397-425 (1977).</li><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+observations+on+hippocampal+neurons+in+dispersed+cell+culture.">Further observations on hippocampal neurons in dispersed cell culture.</a> The Journal of Comparative Neurology. 187 (3), 469-493 (1979).</li><li>Biffi, E., Regalia, G., Menegon, A., Ferrigno, G., Pedrocchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+neuronal+density+and+maturation+on+network+activity+of+hippocampal+cell+cultures:+a+methodological+study.">The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study.</a> PLoS One. 8 (12), e83899 (2013).</li><li>Hiragi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Human+Induced+Pluripotent+Stem+Cell+(hiPSC)-Derived+Neurons+in+Mouse+Hippocampal+Slice+Cultures.">Differentiation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons in Mouse Hippocampal Slice Cultures.</a> Frontiers in Cellular Neuroscience. 11, 143 (2017).</li><li>Zeng, H., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell-type+classification:+challenges,+opportunities+and+the+path+forward.">Neuronal cell-type classification: challenges, opportunities and the path forward.</a> Nature Reviews. Neuroscience. 18 (9), 530-546 (2017).</li><li>Jones, M. R., Villalon, E., Northcutt, A. J., Calcutt, N. A., Garcia, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+myostatin+deficiency+on+motor+and+sensory+axons.">Differential effects of myostatin deficiency on motor and sensory axons.</a> Muscle &amp; Nerve. 56 (6), E100-E107 (2017).</li></ol>

如前所述，PC12及其子克隆被广泛用于研究神经酸盐延伸，因为它们具有优异的转染效率2，3。相比之下，原发神经元的转染率较低，这是通过转染7研究神经质生长调节器的主要障碍。在这里，我们描述了一个方便的协议，用于量化原始神经元中的神经质外生。尽管整体转染效率较低，但超过80%的转染神经元成功地与两种蛋白质共同转?...

神经酸盐，包括斧子和树突，是参与建立神经网络的神经元的投影。神经酸盐的动态生长对神经发育至关重要。然而，下面的基本监管机制仍不清楚。特别是，神经质损伤经常在各种神经退行性疾病和脑损伤后观察到1。因此，研究假定分子在各种神经酸盐生长调节途径中的作用，将增进我们对该过程的理解。此外，它可能揭示各种神经疾病的新治疗目标。神经元细胞系是研究神经元过程的宝贵模型，包括神经质生长，因为它们易于操作和转染2，3。然而，据报道，遗传漂移发生在一些常用的细胞系中，这可能导致其生理反应的变化4。此外，在神经元细胞系和主神经元之间也显示了差异蛋白表达。例如，PC12，一种来自大鼠肾上腺的神经元细胞系，广泛用于研究神经质外生长2，3，不表达NMDA受体5。此外，据提出，与原发神经元相比，小鼠神经母细胞瘤线神经-2a对神经毒素的响应能力降低是由于某些膜受体和电通道6缺乏表达。因此，原神经元是神经质生长研究更可取和更具代表性的模型。然而，主神经元的使用受到其低转染效率7的阻碍。
在这里，我们描述了一种涉及感兴趣蛋白（POI）和EGFP与原发性大鼠皮质神经元共转染的方法。EGFP作为形态标记，用于识别成功转染的神经元，并允许测量神经酸盐。我们使用已报告的化合物/分子来调节神经酸盐生长，验证了这种方法。此外，FE65，一种神经元适配器蛋白，已被证明能刺激神经质生长，用于说明这种方法8，9。该协议涉及 （1） 从胚胎第18天（E18）大鼠胚胎分离原发性皮质神经元，（2）与EGFP和POI（本研究中的FE65）共同转染神经元，以及（3）使用图像处理对神经元的成像和分析软件图像J与NeuronJ插件10，11。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 tweezers</td>
			<td>Regine</td>
			<td>5-COB</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm Circle Cover Slips</td>
			<td>Thermo Scientific</td>
			<td>CB00180RA</td>
			<td>Sterilize before use.</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Invitrogen</td>
			<td>PHZ1063</td>
			<td>Dissolved in DMSO.</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors, 10 cm</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors, 12.5 cm</td>
			<td>World Precision Instruments</td>
			<td>15922</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S302380</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NGF 2.5S Native Mouse Protein</td>
			<td>Gibco</td>
			<td>13257019</td>
			<td></td>
		</tr>
		<tr>
			<td>Nugent Utility Forceps, 10mm, Straight Tip</td>
			<td>World Precision Instruments</td>
			<td>504489</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>pEGFP-C1</td>
			<td>Clontech</td>
			<td>#6084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>pCI FE65</td>
			<td></td>
			<td></td>
			<td>Please see references 8 and 15</td>
		</tr>
		<tr>
			<td>PBS Tablets</td>
			<td>Gibco</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Sigma-Aldrich</td>
			<td>S4147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enhanced green fluorescence protein-based assay for studying neurite outgrowth in primary neurons

神经细胞生长是神经系统发育过程中神经回路形成的基本事件。严重的神经质损伤和突触功能障碍发生在各种神经退行性疾病和与年龄相关的退化。研究调节神经酸盐生长的机制，不仅会为大脑发育过程提供有价值的光，而且还可以揭示这些神经紊乱。由于转染效率低，目前研究特定蛋白质对原生哺乳动物神经元中神经质生长的影响具有挑战性。在这里，我们描述了一种通过与EGFP和感兴趣的蛋白质（POI）共同转染原发性大鼠皮质神经元来研究神经质外生的简单方法。该方法允许通过EGFP信号识别POI转染神经元，从而可以精确确定POI对神经质生长的影响。这种基于EGFP的测定为研究调节神经细胞生长的途径提供了一种方便的方法。

为了测试这种方法，我们使用细胞D和神经生长因子NGF，这已被证明分别抑制和刺激神经质生长14，15，16。使用EGFP转染的神经元的中性粒体长度在使用Cyto D或NGF治疗后被测量。EGFP对神经元的转染效率为2.7%（计算1，068个神经元）。如图1A所示，细胞D以剂量依赖性的方?...

Watch this Scientific Journal Video about An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons at JoVE.com

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

在本报告中，我们描述了一个简单的方案，通过与EGFP和感兴趣的蛋白质共同转染，研究胚胎大鼠皮质神经元中的神经质外生。

增强的绿色荧光蛋白测定，用于研究原神经元的神经质生长

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

转染效率低是确定蛋白质如何重新激活原神经元中神经酸盐生长的主要障碍。我们的协议通过ERFP的共转感染来识别成功的转染细胞，克服了这个问题。EGFP和感兴趣的蛋白质的高共发率确保了测定的准确性。因此，现在需要免疫荧光染色。由于神经性细胞在神经再生过程中出现，该方法可应用于确定脑损伤或神经退行性疾病中受损神经元网络的新靶点。在开始手术之前，将无菌的 18 毫米圆形盖玻片放入 12 孔组织培养板的每个孔中，并在加湿的 37 摄氏度培养箱中，将每毫米 5 微克的聚-D-Lysine 溶液涂上 5 微克，至少一小时。在孵育结束时，吸进聚-D-Lysine溶液，并用无菌水冲洗盖玻片。在胚胎第18天，将从一只怀孕时分的斯普拉格·道利大鼠身上采集的子宫放入10厘米的培养皿中，用小解剖剪刀打开子宫和羊膜囊。使用剪刀去除胎盘，用钳子将整个胚胎移植到含有冰镇PBS葡萄糖的10厘米培养皿中。用剪刀沿着头骨的缝合线切割，用一个小的扁平铲子将胚胎大脑转移到含有新鲜冰冷PBS葡萄糖的10厘米新的培养皿中。使用两个五号钳子和解剖显微镜，将两个脑半球从小脑和脑干分离，并切除脑膜。然后将皮层与脑半球分离，将皮层放在含有新鲜冰冷PBS葡萄糖的15毫升管中。对于初级皮质神经元培养，在II类生物安全柜中，允许皮层在4摄氏度下由重力沉淀5分钟，然后用0.05%胰蛋白素EDTA取代PBS葡萄糖。与轻轻的敲击混合，在37摄氏度的水浴中孵育组织10分钟，每两分钟额外轻轻敲击一次。在孵化结束时，用四毫升的维持介质停止消化，并使用一毫升移液器通过温和的三角分离组织。通过离心沉淀细胞，将颗粒重新悬浮在一毫升的维持介质中，进行第二次离心。在第二个离心后在一毫升新鲜维持介质中重新浓缩形成颗粒，以6.5倍10的浓度将分离的神经元镀到每厘米平方浓度为1毫升的维持介质中，在12井板中每井一毫升的维持介质中。在37摄氏度和5%的二氧化碳下两天后，将EGFP和靶向蛋白质粒DNA和转染试剂分两个独立的1.5毫升管中稀释，然后混合在一起。通过将转染混合物添加到井中，用EGFP结构对细胞进行转染。24小时后，用37摄氏度的PBS清洗细胞，并在室温下用4%的甲醛在PBS中修复10分钟。在孵化结束时，每次洗涤用新鲜 PBS 清洗固定细胞三次，并将最小体积的荧光安装介质添加到每个样品的一个显微镜玻璃幻灯片上。然后小心地将涂层盖玻片转移到安装介质上，样品朝向玻璃滑梯。要成像神经石生长，请在荧光显微镜上选择 40 倍的镜面，然后单击"开始"以捕获每个转染至少 40 个完整 EGFP 阳性神经元的图像。当所有神经素都已成像后，使用 NeuronJ 插件打开 ImageJ 中捕获的图像，并测量每个神经元从细胞体到生长锥顶的最长神经素的长度。然后分析通过软件获得的数据，确定靶蛋白对神经土生长的影响。细胞茶素D抑制神经素延长在剂量依赖的方式，而神经素外生生长增强的神经元治疗神经生长因子。此外，与未转化的神经元相比，神经元生长适配器蛋白FE65显著刺激神经素的生长倍。尽管转染效率较低，但免疫荧光分析显示，超过80%的两天细胞培养可以成功地与EGFP和FE65联合感染。EGFP表达在转染后6小时检测，而FE65在第12小时首次检测到，两个信号在转染后长达7天。不同数量的FE65质粒DNA的原发性大鼠皮质神经元的转染揭示了与0至0.5微克FE65质粒的neurite延长剂量依赖性增加。然而，在通过0.5或1微克质粒转染的神经元中，没有显著的生长差异。在尝试该协议时，重要的是将适当的区域与胚胎大脑分离。该技术还可用于研究人类 iPSC 衍生神经元的神经生长，为再生医学提供了宝贵的工具。

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

将毛细血管纳入内皮细胞珠发芽试验

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Induction and Validation of Cellular Senescence in Primary Human Cells

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

一种对绿色藻原体同步文化进行深入分析的多表率提取方法

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...