Name: stomata tape-peel: an improved method for guard cell sample preparation
Uploaded: 2018-07-15T13:00:00
Description: Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

我们感谢布赫兹数字视频制作团队的丹尼尔. 陈, 协助编辑视频。该研究的气孔保护细胞, 蛋白质组学和新陈代谢在陈实验室得到了资助, 美国国家科学基金会 (0818051, 1158000 和 1412547)。文文香港获中国奖学金委员会支持。Qiuying 博士获得中国奖学金委员会和国家自然科学基金 (31570396) 的支持。

1. 种植植物
<ol><li>在灌封混合物中发芽种子。在生长室种植植物在一个光强度的140µmol 光子 m−2 s −1和光周期8小时在22°c 和 16 h 黑暗在18°c 2 星期。</li>
	<li>将类似大小的移栽苗单独放入4英寸直径的花盆中, 并在步骤1.1 所述相同条件下生长3周。 注: 自来水厂每周两次, 以保持土壤湿润。</li>
</ol>2. 制备富气孔
<ol><li>用手术刀把5周大的叶子移开.</li>
	<l...

<ol>
	<li>Acharya, B., Assmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormone+interactions+in+stomatal+function.">Hormone interactions in stomatal function.</a> Plant Molecular Biology. 69 (4), 451-462 (2009).</li><li>Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., Waner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+cell+signal+transduction.">Guard cell signal transduction.</a> Annual Review of Plant Physiology and Plant Molecular Biology. 52, 627-658 (2001).</li><li>Obulareddy, N., Panchal, S., Melotto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guard+Cell+Purification+and+RNA+Isolation+Suitable+for+High-Throughput+Transcriptional+Analysis+of+Cell-Type+Responses+to+Biotic+Stresses.">Guard Cell Purification and RNA Isolation Suitable for High-Throughput Transcriptional Analysis of Cell-Type Responses to Biotic Stresses.</a> Molecular Plant-Microbe Interactions. 26 (8), 844-849 (2013).</li><li>Pandey, S., Wang, X. Q., Coursol, S. A., Assmann, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+applications+of+Arabidopsis+thaliana+guard+cell+protoplasts.">Preparation and applications of Arabidopsis thaliana guard cell protoplasts.</a> New Phytologist. 153 (3), 517-526 (2002).</li><li>Schroeder, J. I., Raschke, K., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+Dependence+Of+K++Channels+In+Guard-Cell+Protoplasts.">Voltage Dependence Of K+ Channels In Guard-Cell Protoplasts.</a> Proceedings of the National Academy of Sciences of the United States of America. 84 (12), 4108-4112 (1987).</li><li>Wu, F. H., 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=Tape-Arabidopsis+Sandwich+-+a+simpler+Arabidopsis+protoplast+isolation+method.">Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method.</a> Plant Methods. 5, 10 (2009).</li><li>Svozil, J., Gruissem, W., Baerenfaller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+targeting+of+proteins+in+Arabidopsis+leaf+mesophyll,+epidermal+and+vascular+tissues.">Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.</a> Frontiers in Plant Science. 6, (2015).</li><li>Schneider, C., Rasband, W., Eliceiri, K. <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>Agnew, B., Murray, D., Patton, W. <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+solid-phase+fluorescence-based+protein+assay+for+quantitation+of+protein+electrophoresis+samples+containing+detergents,+chaotropes,+dyes,+and+reducing+agents.">A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents.</a> Electrophoresis. 25 (15), 2478-2485 (2004).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> Bio-protocol. Bio101 (80), (2011).</li><li>Thompson, 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=Tandem+mass+tags:+A+novel+quantification+strategy+for+comparative+analysis+of+complex+protein+mixtures+by+MS/MS+(vol+75,+pg+1895).">Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS (vol 75, pg 1895).</a> Analytical Chemistry. 78 (12), 4235-4235 (2006).</li><li>Wiese, S., Reidegeld, K. A., Meyer, H. E., Warscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+labeling+by+iTRAQ:+A+new+tool+for+quantitative+mass+spectrometry+in+proteome+research.">Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry in proteome research.</a> Proteomics. 7 (3), 340-350 (2007).</li></ol>

保护细胞是研究植物信号转导机制的模型系统, 重要的是要确保研究中使用的样品的制备最适合回答生物学问题。尽管植物研究界对保护细胞的兴趣越来越浓厚, 但对于如何制备气孔保护细胞, 使气孔运动和生理以及分子变化在一个研究中都没有普遍的方法系统。隔离它们的挑战在很大程度上归因于小规模、低丰度和独特的结构, 这可以使它们或多或少难以从整个树叶中移除。
...

气孔在植物的整体生理和健身中起着重要的作用。这些微小的植物特定的结构是由两个专门的表皮细胞组成的, 称为守卫细胞, 在叶的背面最丰富的发现。气孔是在植物和大气之间交换气体和通过蒸腾控制水分损失所必需的。保护细胞通过不同外部 (例如, 光, 湿度, 病原体接触, 二氧化碳水平) 和内部 (e., 内源激素) 的整合来调节气孔孔径,刺激 1,2。在过去的20年中, 这种细胞类型已经成为研究植物细胞信号过程的模型系统。这些细胞构成了叶中总细胞的一小部分;因此, 以单细胞的方式研究其独特的细胞、生物化学和分子特性, 就有必要将它们与其他叶细胞类型分离。在过去, 守卫细胞研究经常介入了卫兵细胞原生质体的准备 (GCP)3,4,5。这通常需要使用大量的细胞壁降解酶和或通过混合的机械干扰, 并证明是昂贵和耗时, 因为它通常需要很多小时来准备一个 GCP 样品, 往往是很少的产量。更重要的是, 由于保护细胞作为完全对调节气孔孔径, 使用 GCP 可以被视为一个人工系统, 研究保护细胞信号。
在这里, 我们开发了一种方法来制备气孔, 其中完整的保护细胞丰富和保持生理反应的刺激。这种方法的灵感来自于分离叶肉细胞原生质体6,7的方法。在我们的方法, 气孔是准备使用透明的透明胶带, 首先分离的大部分叶肉细胞的正面层的叶子从背面层包含路面和守卫细胞。这是通过贴一块胶带到叶子的两侧, 并剥离它们分开。在气孔开放的缓冲区中, 允许从背面层的细胞恢复到光下。后来, 其余的路面细胞被删除使用少量的细胞壁降解酶, 留下保护细胞, 丰富的磁带。
在这种简单的方法中, 气孔保护细胞既可行又反应灵敏, 可以快速有效地制备, 花不到一小时就能从50皮中获得丰富的气孔保护细胞, 成本最低。这里的方法对植物细胞的损伤比以前的原生质体制备方法少。此外, 从这种方法收集的材料产生理想的蛋白质量。最重要的是, 由于叶子不受混合或长时间的消化, 并且保护细胞保持不变, 研究结果将更接近于保护细胞的自然生物学。利用这种方法, 气孔运动可以与分子水平的变化实时相关。因此, 从研究中获得的知识使用这种制备方法, 对于更全面地了解防护细胞信号是非常重要的。
我们用植物拟南芥作为模型;但该协议可应用于其他植物中的气孔保护细胞的富集, 如甘蓝型油菜的消化时间小改性。作为概念的证明, 我们已经证明, 通过这种方法丰富的气孔保护细胞是可行的, 并响应的刺激, 如荧光素二乙酸 (FDA) 的研究, 并利用植物激素脱落酸 (ABA), 分别。此外, 我们已经证明, 高质量的蛋白质可以从这些守卫细胞分离。在这里, 我们描述了这个过程的详细协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:889px;" width="889">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Stainless Steel Surgical Scalpel</td>
			<td style="width:297px;">Feather&#160;</td>
			<td style="width:119px;">&#160;NC9999403</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Scotch Tape (3M)</td>
			<td style="width:297px;">ULINE</td>
			<td style="width:119px;">S-9781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Petri Dish</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td>BR455701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase (Onozuka R-10)&#160;</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macerozyme R-10</td>
			<td>Yakult Pharmaceutical Industry Co., Ltd.</td>
			<td>21560003-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DM6000B Microscope&#160;</td>
			<td>Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease and Phosphatase Inhibitor Cocktails</td>
			<td style="width:297px;">Thermo Fisher Scientific&#160;</td>
			<td>&#160;PI78443</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Oak Ridge High-Speed PPCO Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>3119-0030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZQ Protein Quantitation Kit</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>R33200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescein Diacetate (FDA)</td>
			<td style="width:297px;">Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Abscisic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Metro Mix 500</td>
			<td style="width:297px;">BWI Companies&#160;</td>
			<td style="width:119px;">TX-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Laemmli Sample Buffer</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Bio-Safe Comassie (G-250)</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">161-0786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microcentrifuge Tube (2mL)</td>
			<td style="width:297px;">USA Scientific, Inc.</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">ImageJ software</td>
			<td style="width:297px;">National Institute of Health</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Tweezers&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4017-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">12% Mini-PROTEAN TGX Precast Gel</td>
			<td style="width:297px;">Bio-Rad</td>
			<td style="width:119px;">456-1043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slides</td>
			<td style="width:297px;">Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
	</tbody>
</table>

stomata tape-peel: an improved method for guard cell sample preparation

保护细胞的研究对于了解这种细胞类型对植物整体功能的特殊贡献是至关重要的。然而, 通常很难分离, 因此研究它们往往会带来挑战。
本研究建立了气孔保护细胞的富集方法。该协议利用透明胶带分离出保护细胞并从细胞中提取蛋白质。该方法在隔离过程中提高了保护细胞的完整性和产量, 为细胞信号过程的研究以及它们与气孔运动表型的相关性提供了可靠的样本。在这种方法中, 植物叶被分成两部分的胶带和剥离分开, 以消除背面的一侧。本协议适用于拟南芥叶组织分离保护细胞。用荧光素双乙酸 (FDA) 对细胞进行治疗, 以确定活性和脱落酸 (aba), 以评估 aba 对气孔运动的反应。最后, 该协议对制备富含气孔保护细胞和分离蛋白质具有重要意义。这里获得的细胞和蛋白质的质量使生理学和其他生物研究得以实现。

图 1显示了拟南芥保护细胞在叶肉和表皮细胞消化前后的代表性图像。使用 FDA 测量酶活性和细胞膜完整性 (图 1B和1D) 可以观察到叶肉和表皮细胞去除前后的保护细胞活力。图 2说明拟南芥的气孔运动对 ABA 的治疗反应。与 ABA 治疗30分钟后气孔孔径减少50% 以上。图 2A是气?...

Watch this Scientific Journal Video about Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation at JoVE.com

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

本协议描述了一种制备富含气孔保护细胞的方法, 用于生理学和其他生物研究。

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Research

JoVE Journal

Biology

MALDI Sample Preparation: the Ultra Thin Layer Method

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Western Blotting: Sample Preparation to Detection

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Sample Preparation for Mass Cytometry Analysis

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...