Name: real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line
Uploaded: 2014-08-09T15:00:00
Description: Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1，准备工作<ol><li>将实时细胞分析仪的台在组织培养箱设定为37℃，并用5％的CO 2。完成所有的细胞培养操作和药物治疗在无菌条件下，组织培养罩。 </li><li>注：神经元细胞系中，需要不同的温度设置进行比较，以对培养的标准条件下，应作相应的调整。对于弱粘附细胞系，用包衣剂，以促进细胞的金微电极中的电子板96的孔的底部的相互作用。 </li><li>制备1,000倍原液的药理化合物用于?...

<ol>
	<li>Cavallucci, V., D'Amelio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matter+of+life+and+death:+the+pharmacological+approaches+targeting+apoptosis+in+brain+diseases.">Matter of life and death: the pharmacological approaches targeting apoptosis in brain diseases.</a> Curr. Pharm. Des. 17 (3), 215-229 (2011).</li><li>Bull, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+CHaPD+001:+Review+of+Environmental+Chemicals+and+Neurotoxicity.">HPA CHaPD 001: Review of Environmental Chemicals and Neurotoxicity.</a> Focus on Neurological Diseases. , (2007).</li><li>Ke, N., Wang, X., Xu, X., Abassi, Y. 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+xCELLigence+system+for+real-time+and+label-free+monitoring+of+cell+viability.">The xCELLigence system for real-time and label-free monitoring of cell viability.</a> Methods Mol. Biol. 740, 33-43 (2011).</li><li>Limame, R., 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=Comparative+analysis+of+dynamic+cell+viability,+migration+and+invasion+assessments+by+novel+real-time+technology+and+classic+endpoint+assays.">Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.</a> PLoS One. 7 (10), e46536 (2012).</li><li>Diemert, S., 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=Impedance+measurement+for+real-time+detection+of+neuronal+cell+death.">Impedance measurement for real-time detection of neuronal cell death.</a> J. Neurosci. Methods. 203 (1), 69-77 (2012).</li><li>Kustermann, S., 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=A+label-free,+impedance-based+real+time+assay+to+identify+drug-induced+toxicities+and+differentiate+cytostatic+from+cytotoxic+effects.">A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects.</a> Toxicol In Vitro. 27 (5), 1589-1595 (2013).</li><li>Marinova, Z., Walitza, S., Gr&#252;nblatt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT2A+serotonin+receptor+agonist+DOI+alleviates+cytotoxicity+in+neuroblastoma+cells:+role+of+the+ERK+pathway.">5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: role of the ERK pathway.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 44, 64-72 (2013).</li><li>Gonz&#225;lez-Maeso, 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=Hallucinogens+recruit+specific+cortical+5-HT(2A)+receptor-mediated+signaling+pathways+to+affect+behavior.">Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., Nakata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+(5-HT)+induces+glial+cell+line-derived+neurotrophic+factor+(GDNF)+mRNA+expression+via+the+transactivation+of+fibroblast+growth+factor+receptor+2+(FGFR2)+in+rat+C6+glioma+cells.">Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells.</a> J. Neurochem. 106 (1), 244-257 (2008).</li><li>Galante, 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=Differential+toxicity,+conformation+and+morphology+of+typical+initial+aggregation+states+of+A&#946;1-42+and+A&#946;py3-42+beta-amyloids.">Differential toxicity, conformation and morphology of typical initial aggregation states of A&#946;1-42 and A&#946;py3-42 beta-amyloids.</a> Int. J. Biochem. Cell. Biol. 44 (11), 2085-2093 (2012).</li><li>Sheline, C. T., Cai, A. L., Zhu, J., Shi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+or+target+deprivation-induced+neuronal+death+causes+oxidative+neuronal+accumulation+of+Zn2+and+loss+of+NAD.">Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+and loss of NAD.</a> Eur. J. Neurosci. 32, 894-904 (2010).</li></ol>

当前协议提出了一种实时的细胞分析仪的实用程序，在连续和无标记的条件下评估的化合物在神经元细胞系中的神经保护/神经毒性作用，并深入了解所涉及的效果的第二信使途径。 即使在实时细胞分析仪的实用程序来研究细胞毒性和药物对细胞增殖的影响一般认为，只有很少的研究已经在神经元相关的细胞类型中使用它。我们先前的研究显示了实时细胞分析仪的实用程序来?...

神经细胞死亡起到许多脑相关疾病1的病理生理学中起关键作用。的可靠和高通量的体外毒性试验的可用性是至关重要的，以获得更好的洞察神经毒性的作用机制并帮助选择神经保护分子作为治疗候选药物开发2。不过，也有不少限制使用最广泛的体外神经毒性assays.They评估神经毒性/神经保护在单一时间点不让动力学拆分;经常使用的标签或探针可与信号通路干扰，并在同一细胞群限制进一步的研究，并且通常是劳动密集型的，并且在许多情况下，不提供机械性的认识。在本研究中我们展示了一个实时的阻抗为基础的细胞分析仪的实用程序来确定神经毒性和神经保护作用的实时，并在神经元细胞系无标签条件，并通过所涉及到的作用的第二信使途径的分析洞察下游机制。 先前的研究已经证实了实时细胞分析的有效性，以确定在标准技术3,4,5,6对比的细胞毒性以及对细胞系的细胞增殖的影响。例如，在标准细胞存活力WST-1法的读数与细胞指数值之间观察到几个时间点下基底扩散条件和HeLa细胞3两种不同的有毒范式后的良好相关性。在A549和MDA-MB-231细胞增殖和细胞毒性挑起与微管 ​​稳定剂紫杉醇表现出非常相似的值时，通过细胞指数测量，评估和标准地使用磺酰罗丹明B（SRB）法4。在永生的海马神经元的神经细胞系HT-22细胞指数测量，验证了自己的能力吨ö检测细胞增殖，谷氨酸毒性和细胞保护作用对广泛使用的3（4,5 -二甲基-2-基） - 2,5 -二苯基溴（MTT）法5。在同一研究中的MTT法检测结果和细胞指数测量也由泛Caspase抑制剂QVD 5个测量神经元祖细胞的增殖，细胞毒性后的生长因子剥夺和细胞毒性的救援很好的相关性。细胞毒性通过凡德他尼（血管内皮生长因子受体和表皮生长因子受体抑制剂）诱导的NIH 3T3细胞显示出与细胞指数值或中性红摄取试验6测定结果相似。 我们最近使用的实时细胞分析系统，以评估血清素2A的神经保护作用（5-HT 2A）受体激动剂（±）-2,5 -二甲氧基-4-iodoamphetamine盐酸盐（DOI）的神经元细胞系（ SK-N-SH细胞），并筛选山高的参与通过监测上观察到的神经保护7的化学抑制的效果第二信使通路。有趣的是，对5-HT 2A受体具有两个致幻和nonhallucinogenic激动剂（如DOI和麦角乙脲，分别），其可以激活两个共同和不同的第二信使通路8。 所提出的技术的优点是，它允许收集在天的过程中对细胞存活的实时信息，划定第二信使途径参与其中，以评估增殖效应的神经保护作用的可能贡献，并选择一个最佳的时间为在相同的细胞群附加端点研究。在当前的协议的工作流程的示意图示于图1。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

许多大脑相关的疾病具有参与其病理生理学的神经元细胞死亡。改进的体外模型来研究药物和涉及将有助于深入了解神经保护/神经毒性的分子机制和可能潜在地促进药物开发下游途径的神经保护或神经毒性作用。然而，许多现有的体外毒性实验有很大的局限性-大多数评估神经毒性和神经保护在一个单一的时间点，而不是让观察的时间过程和效果动力学。此外，机会来收集关于涉及神经保护中的实时下游信号传导途径的信息是非常重要的。在当前的协议中，我们描述了使用实时的基于阻抗的细胞分析装置，以确定血清素2A（5-HT 2A）受体激动剂在神经元细胞系在无标记和实时康迪特治疗作用使用阻抗测量离子。此外，我们证明，第二信使途径的抑制剂可用于描绘参与神经保护作用的下游分子。我们还描述了该技术的实用程序来确定对细胞增殖的效果，是否有助于所观察到的保护作用。该系统采用称为E-平板的包含交替的孔的底部表面的金微电极阵列上，作为细胞传感器专用微电子板。阻抗读出是通过贴壁细胞，细胞生存力，形态和粘附的数量进行修改。一种称为细胞指数无量纲参数是从所述电阻抗测量得到和用于表示该细胞的状态。总体而言，实时基于阻抗的细胞分析装置允许进行实时，神经保护和神经毒性的无标记的评估，以及第二信使途径参与了评价，有助于更在体外的潜在的神经保护化合物的详细的和高通量的评估，选择治疗的候选人。

<html> 血清剥夺导致在细胞指数值，它可以连续地实时细胞分析仪进行监测，以减少 神经刺激的细胞导致的降低细胞指数值，它可在实时的呈现技术和动态这是依赖于特定的神经刺激和研究的细胞类型进行监测。 图2显示了增加的当SK-N-SH细胞生长在增殖培养基中，直到达到平台（绿线）和细胞指数的下降之后稳定在新的水平较低的开关的细胞血清剥夺...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

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