Name: assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture
Uploaded: 2017-05-10T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

浙江省自然科学基金（LY15H310007），浙江省非营利技术应用研究项目（2016C37110），国家自然科学基金（U1503223,81673407），宁波国际科学和技术合作项目（2014D10019），宁波市生命科学与卫生创新队（2015C110026），广东省国际科技合作项目（2013B051000038），深圳基础研究计划（JCYJ20160331141459373），广东红香港技术合作资助计划（GHP / 012 / 16GD），香港研究资助局（15101014），香港理工大学（G-YBGQ）和宁波大学黄金大学基金。

所有程序均遵循国家卫生研究院（NIH）实验动物护理和使用指南（NIH出版物第80-23号，修订1996年），并得到宁波大学体育动物护理与使用委员会（IACUC）的批准（ SYXK-2008-0110）。 解决方案和文化媒体的准备注意：试剂和库存需要在无菌条件下制备。通过使用孔径为0.22μm的过滤器进行过滤灭菌。 <ol><li>通过向10mL双蒸水（ddH 2 O）中加入5mg的PLL制备?...

<ol>
	<li>Yu, J., 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=Indirubin-3-oxime+prevents+H2O2-induced+neuronal+apoptosis+via+concurrently+inhibiting+GSK3beta+and+the+ERK+pathway.">Indirubin-3-oxime prevents H2O2-induced neuronal apoptosis via concurrently inhibiting GSK3beta and the ERK pathway.</a> Cell Mol Neurobiol. , (2016).</li><li>Cui, 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=The+anti-cancer+agent+SU4312+unexpectedly+protects+against+MPP(+)+-induced+neurotoxicity+via+selective+and+direct+inhibition+of+neuronal+NOS.">The anti-cancer agent SU4312 unexpectedly protects against MPP(+) -induced neurotoxicity via selective and direct inhibition of neuronal NOS.</a> Br J Pharmacol. 168 (5), 1201-1214 (2013).</li><li>Jebelli, J., Piers, T., Pocock, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Depletion+of+Microglia+from+Cerebellar+Granule+Cell+Cultures+Using+L-leucine+Methyl+Ester.">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J Vis Exp. (101), e52983 (2015).</li><li>Labno, 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> Two way to count cells with ImageJ. , (2008).</li><li>Haag, D., 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=Nos2+Inactivation+promotes+the+development+of+medulloblastoma+in+Ptch1(+/-)+mice+by+deregulation+of+Gap43-dependent+granule+cell+precursor+migration.">Nos2 Inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.</a> Plos Genet. 8 (3), e1002572 (2012).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), e990 (2009).</li><li>Messer, 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+maintenance+and+identification+of+mouse+cerebellar+granule+cells+in+monolayer+culture.">The maintenance and identification of mouse cerebellar granule cells in monolayer culture.</a> Brain Res. 130 (1), 1-12 (1977).</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=Novel+dimeric+acetylcholinesterase+inhibitor+bis7-tacrine,+but+not+donepezil,+prevents+glutamate-induced+neuronal+apoptosis+by+blocking+N-methyl-D-aspartate+receptors.">Novel dimeric acetylcholinesterase inhibitor bis7-tacrine, but not donepezil, prevents glutamate-induced neuronal apoptosis by blocking N-methyl-D-aspartate receptors.</a> J Biol Chem. 280 (18), 18179-18188 (2005).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1 (5), 2406-2415 (2006).</li><li>Cheung, G., Cousin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+synaptic+vesicle+pool+replenishment+in+cultured+cerebellar+granule+neurons+using+FM+Dyes.">Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM Dyes.</a> J Vis Exp. (57), e3143 (2011).</li><li>Atlante, A., 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=Genistein+and+daidzein+prevent+low+potassium-dependent+apoptosis+of+cerebellar+granule+cells.">Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells.</a> Biochem Pharmacol. 79 (5), 758-767 (2010).</li><li>Silva, R., Rodrigues, C., Brites, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+cultured+neuronal+and+glial+cells+respond+differently+to+toxicity+of+unconjugated+bilirubin.">Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin.</a> Pediatr Res. 51 (4), 535-541 (2002).</li><li>Jekabsons, M. B., Nicholls, D. 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该协议是从先前已经描述过的程序6,7修改的。研究人员花费时间尝试通过在体外培养原代神经元时改善条件来减少非神经元细胞生长8 。然而，即使改善的培养条件，一些胶质细胞仍然存在。此外，非神经元细胞在原代神经元培养中是必需的，因为它们有助于神经元生长和成熟9 。在这项研究中，Ara-C被用来尽可能减少神经胶质细胞的生长，尽管在CGN培养?...

通常使用比色细胞毒性测定法（例如3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四唑溴化物（MTT）测定法来测量体外细胞活力。来自大鼠的原代培养的小脑颗粒神经元（CGN）对各种神经毒素敏感，包括1-甲基-4-苯基吡啶鎓离子，过氧化氢和谷氨酸盐1,2 。因此，CGN培养可用作神经科学领域的体外模型。 CGN培养物可以含有多种细胞，包括神经元和神经胶质细胞，其可占CGN培养物中总细胞的约1％。然而，与神经元相比，神经胶质细胞对神经毒素的反应不同，导致通过比色测定法测量的神经元存活力的偏差3 。 在活细胞中，荧光素二乙酸酯（FDA）可以通过酯酶转化为荧光素。碘化丙啶（PI）可以在穿透死细胞后与DNA相互作用，可用于指示培养物中的凋亡。因此，FDA-PI双染可以同时评估活细胞和死细胞，这表明通过组合比色法可以更精确地测量细胞活力。此外，通过添加Hoechst蓝色荧光染色剂可以进一步提高细胞活力的准确性。本文提出的方案描述了FDA-PI双染色和FDA-PI-Hoechst三重染色，可用于准确分析原代培养的CGNs中的神经元活力。 该协议利用其不同尺寸和形状可视化和区分CGN和胶质细胞。染色后，从通过荧光显微镜拍摄的代表性图像中计数活神经元和死亡神经元的数量。通过比较典型的CGN t来排除大尺寸胶质细胞在荧光模式下，在相位对比模式下拍摄。可以进行类似的策略来测量含有神经元和神经胶质细胞的混合细胞培养物中的神经元活力，例如原代皮层培养物和海马培养物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-Arabinofuranoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099141&#160;</td>
			<td>high quality FBS is essential for culture</td>
		</tr>
		<tr>
			<td>100&#215; glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#215; anti-biotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BME medium</td>
			<td>Gibco</td>
			<td>21010046&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Yesen</td>
			<td>40731ES10</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit Anti-GAP43 antibody</td>
			<td>Abcam</td>
			<td>ab75810</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse Anti-GFAP antibody</td>
			<td>Cellsignaling</td>
			<td>3670</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sangon Biotech</td>
			<td>A602440</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>T4665</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse&#160;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Volac</td>
			<td>Z310727</td>
			<td>burn&#160; the tip round before use</td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707783</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707759</td>
			<td>high quality cell culture plate&#160; is essential for culture</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 5 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissect microscope</td>
			<td>Shanghai Caikang</td>
			<td>XTL2400</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td>311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td>TI-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter and emmision cubes</td>
			<td>Nikon</td>
			<td>B-2A, G-2A, UV-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo software</td>
			<td>Nikon</td>
			<td>NIS-Elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics editor softeware</td>
			<td>Adobe</td>
			<td>Photoshop CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Image process softeware</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture

原代培养的小脑颗粒神经元（CGNs）已广泛用作神经科学和神经药理学研究中的体外模型。然而，CGN培养中神经胶质细胞和神经元的共存可能导致精确评估神经元活力的偏差。荧光素二乙酸酯（FDA）和碘化丙啶（PI）双染法已被用于通过同时评估活细胞和死细胞来测量细胞活力。我们使用FDA-PI双染来提高比色测定的敏感性，并评估CGNs中的神经元活力。此外，我们添加蓝色荧光DNA污渍（ 例如 Hoechst）以提高准确性。该协议描述了如何通过在CGN培养中使用这些方法来提高神经元活力评估的准确性。使用该方案，可以通过使用荧光显微镜来排除胶质细胞的数量。可以应用类似的策略来区分不想要的gli来自各种混合细胞培养物中的神经元的细胞，例如原代皮质培养物和海马培养物。

GAP43（红色）和GFAP（绿色）的双重免疫染色分别用于分析神经元和神经胶质细胞的形状。神经元和神经胶质细胞均存在于CGN培养物中。 GFAP阳性神经胶质细胞大，形状不规则，如图像中的箭头所示（ 图1 ）。当用于测量神经元活力时，细胞活力的传统测定法不能区分神经元神经元。因此，可以区分胶质细胞与神经元的FDA-PI和FDA-PI-Hoechst染色有利于准确评估CGN培...

Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

该协议描述了如何使用荧光素二乙酸酯（FDA）和碘化丙啶（PI）双重染色在培养的小脑颗粒神经元中准确测量神经元活力，这是一种初级神经元培养物，用作神经科学和神经药理学研究中的体外模型。

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

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