我们非常感谢马修罗伯特韦瑟优质的技术服务和约翰拉尔夫，威斯康星大学的宝贵意见，讨论和杨木样本。这项工作是由化学，地球科学和生物科学部，基础能源科学，美国能源部科学办公室（奖励没有DE - FG02 - 91ER20021）和大湖的美国能源部（DOE）的办公室生物能源研究中心（美国能源部的BER办公室科学DE - FC02 - 07ER64494）。

1。细胞墙隔离<ol><li>研磨约60 - 70mg的空气，或冷冻干燥与5.5毫米，在2毫升sarstedt使用retschmill（1分钟，25赫兹）的螺丝帽管不锈钢球的植物材料。另一种方法，使用一种高通量的研磨和配药机器人称为iWall是我 3部分。 </li><li>删除，然后再继续与细胞壁分离过程的钢球 细胞壁物质准备详细的协议是在我3部分所示。为了完整的书面协议的步骤。 </li><li>加1.5毫升70％的含水乙醇，旋涡彻底</li><li>以10,000 rpm离心10分钟沉淀不溶于酒精残留</li><li>吸出或倒出上清液</li><li>添加1.5毫升氯仿/甲醇（1:1 V / V）的解决方案去渣，动摇管，彻底悬浮颗粒</li><li>离心10分钟的转速为10,000和吸出或倒出上清液</li><li>重新悬浮颗粒的丙酮在500 UL </li><li>溶剂蒸发与空气流在35 ° C，直到干 如果需要干燥的样品可存放在室温，直到进一步的处理。 </li><li>要开始重新悬浮颗粒在一个0.1M的sodiumacetate缓冲pH值5.0 1.5毫升样品的淀粉去除。 </li><li>第20分钟sarstedt管和热。在80 ° C在加热块。 </li><li>暂停在冰上冷却</li><li>添加下面的代理商颗粒：35微升0.01％Sodiumazide（南3），35μL淀粉酶（50μg/1mLH 2 O，芽孢杆菌的物种，西格玛）; 17μL普鲁兰（18.7芽孢杆菌acidopullulyticus单位;西格玛） 。第管和涡彻底。 </li><li>悬挂在37℃摇床中的C孵育过夜。定向管水平助手改善混合。 </li><li>热悬浮在100 ° C下10分钟，在加热块终止消化。 </li><li>离心机（10,000 RPM，10分钟），并丢弃上清液含溶解的淀粉</li><li>洗，加入1.5毫升的水，振荡，centriguation，洗涤水调迁，其余颗粒三次。 </li><li>重新悬浮颗粒的丙酮在500 UL </li><li>蒸发与干燥空气流在35 ° C，直到溶剂。这可能是必要的，也打破了管具有更好的干燥锅铲的材料。 干燥后的材料呈现孤立的细胞壁（lignocellulosics）。如果需要干燥的样品可存放在室温，直到进一步的处理。 </li></ol> 2。矩阵多糖成分这种方法实质上是一种修改Albersheim 1发表方法。 <ol><li>要确定墙体材料的单糖组成，重达2毫克2毫升starstedt管细胞进入墙体材料通过手工或使用iWall，机器人机器人研磨和称重一个highthroughput时尚。 </li><li>新增20肌醇解决方案，作为内部标准（5mg/ml）UL。我们建议对于2毫克细胞壁样品中添加100微克。 </li><li> 250 UL丙酮冲洗管壁收集试管底部的细胞壁物质，丙酮蒸发下的气流非常温柔。 </li><li>对于弱的酸水解2M三氟乙酸（TFA）每个样品中加入250 UL。添加TFA的仔细，以确保没有物质泼到管壁。 </li><li>第紧密孵育90分钟，在121 ° C在加热块。 </li><li>冷却加热块上的冰和样品。 </li><li> 10分钟10000转离心管。 </li><li>包含派生的单糖，以确保不打扰颗粒材料的玻璃螺丝帽小瓶矩阵多糖的酸性上清转移100 UL。颗粒可以用下面的结晶纤维素测定（见3。） </li><li>蒸发装置中的温柔下的空气流蒸发TFA在玻璃管。 </li><li>加入300μl2 - 丙醇，旋涡和蒸发在25℃（共3次重复） </li><li> alditol醋酸衍生过程的第一步是执行其相应的alditols减少单糖。为此，添加200μL一个硼氢化钠溶液每个干燥样品。准备一个新鲜的解决方案，每次使用10毫克每1M的氢氧化铵1毫升，钠硼氢化。 </li><li>离开玻璃小瓶，在室温下为1.5小时</li><li>中加入冰醋酸150 UL的解决方案</li><li>涡和蒸发在25℃。 </li><li>加250μL醋酸酸/甲醇（1:9，V / V），旋涡和蒸发在25˚彗星</li><li>加250μL甲醇，根据气流和蒸发（共3次重复） </li><li>对于乙酰的alditols，添加50μL醋酸酐和50μL吡啶国家统计局，涡孵育20分钟，在121 ° C在加热块。 </li><li>冰酷样品块，而等待温度降低到接近室温。 </li><li>温柔流在室温下的空气中挥发的试剂。小心：alditol醋酸极易挥发。 </li><li>加入200μl甲苯和在空气中蒸发（X3） </li><li>在最后的步骤alditol醋酸提取。首先，添加500 UL，醋酸乙酯和漩涡掉以轻心。 </li><li>添加2毫升的水，盖管和旋涡。 </li><li> 5分钟2000转离心管获得明确的独立的层（醋酸乙酯，顶部底部的水） </li><li>移液器50 UL与插入瓶的GC / MS的醋酸乙酯层。 </li><li>加入到GC小瓶和第100丙酮UL稀释。样品体积和稀释量可调节，以避免超载的GC / MS，如果样品浓度过高。 可存放于4℃，如果GC / MS分析不立即进行GC -小瓶</li><li>样品注入配备一个四极杆质谱，气相色谱，但火焰离子化检测器也适用。一个Supelco公司SP - 2380（30毫米x0.25毫米× 0.25微米薄膜厚度）列是一个4分钟的溶剂延迟和1.5ml/min的流速。注入样品经过下列程序升温：初始保持在160 ° 2分钟，C，20 ° C /分钟的斜坡至200℃，保持5 min; 20 ° C /分钟的斜坡至245 ° C和举行12分钟;秒杀到270 ° C和举行前5分钟冷却的初始温度为160 ° C 2.26）峰是由大规模的配置文件和/或标准的保留时间确定。单糖是量化的标准曲线。 </li></ol> 3。结晶纤维素含量这种方法实质上是Updegraf 8。有此过程的起始材料的数量：分离细胞墙体材料（见1）或墙体材料已经与2M TFA的（见2.8）要么其余颗粒酸处理后立即的（见2.8）或TFA的治疗颗粒，已与2 - 丙醇和干洗净。 <ol><li>添加TFA的颗粒螺丝皑皑的玻璃试管1毫升的Updegraff试剂（冰醋酸：硝酸：水，8时01分02秒V / V）。 </li><li>第管得紧紧的，旋涡，热在加热块在100 ° C为30分钟。由于这种治疗方法的结果仍然不溶性的颗粒结晶纤维素。 </li><li>在凉爽的块的样本在冰上室温或冷却器</li><li> 15分钟10000转的离心机样本</li><li>弃上清，确保颗粒是不是感到不安，并没有从颗粒物质被删除。为了这个目的，离开约。 150 UL上清管。 </li><li>加入1.5毫升的水，摇匀，离心，弃上清做上述</li><li>重复洗涤过程3，使用丙酮1.5毫升的额外时间</li><li>空气干燥颗粒，轻轻地与空气，或让替补席上干通宵</li><li>什么是所谓的Saeman水解，沉淀（结晶纤维素）现在已经完全水解成葡萄糖。为此sarstedt管加175μL72％硫酸</li><li>在室温下孵育30分钟，旋涡和另外15分钟的孵育</li><li>添加825μl水和涡</li><li> 5分钟10000转的离心机样本。可能有一些棕色的不溶性物质，木质素，留在管。 </li><li>上清的葡萄糖含量测定采用比色法蒽酮测定。这个实验是在96孔聚苯乙烯酶标板。 </li><li>作标准曲线使用1mg/ml的葡萄糖股票（存储在0 ° C）和创建重复吹打0，2，4，6，8，和10 UL，0，2，4，6，8，和10微克的标准成单独的适当以及。填写每个水以及高达100 UL。 </li><li>添加到单独的标准相同的微孔板细胞，但每个样品上清液10μL和90毫升的水。 </li><li>加200μL新鲜配制的蒽酮试剂（蒽酮溶解在浓硫酸，蒽酮2毫克/毫升硫酸） </li><li>加热板为30分钟，在80℃（铝散热器）在烤箱。含葡萄糖的样品依次从黄到蓝绿色。 </li><li>让冷却到室温的板，彻底动摇。 </li><li>在625毫米，使用酶标仪读板吸收。 </li><li>葡萄糖（因而结晶纤维素含量）计算吸光度标准曲线建立的同一个盘子。 </li></ol> 4。代表性的成果墙上分析的一个例子是在图2。在这种情况下，杨树干（木）协议部分所述的各种程序进行了分析。矩阵宝lysaccharide成分是通过一个例子来确定在植物细胞壁中的典型糖目前的色谱中强调，岩藻糖，鼠李糖，木糖，阿拉伯糖，半乳糖，甘露糖和葡萄糖（内部标准肌醇）。杨树的半纤维素主要成分是木聚糖，木糖含量高表明。然而，这些糖类丰富，会有所不同取决于原料used4。在这种分析中的葡萄糖是来自半纤维素xyloglucan和无定形纤维素。由于分析的数据可以作为MOL％或微克/毫克墙体材料（或干重）。结晶纤维素的内容是不言自明，可以预期值壁干重的20-50％之间。杨树木材的木质纤维组成，根据在这里提出，并在部分的I3的结果是21％的木质素，30％的半纤维素和41％的结晶纤维素。其余部分将灰。 <img src="/files/ftp_upload/1837/1837fig1.jpg" alt="图1" /> 图1：木质纤维分析概述。细胞壁（lignocellulosics）原油干植物材料隔离。墙体材料，然后加权成等份，并细分为各种检测。成立后，治疗用弱酸（2M TFA），墙体材料，alditol醋酸衍生产生的可溶性单糖，并通过GC - MS分析矩阵多糖成分。弱酸处理的残留物洗净，用所谓的Updegraff试剂背后只留下的不溶性crystlline纤维素。纤维素溶解的硫酸和由葡萄糖含量的测定比色法确定量化。同时，木质素的内容和组成可以判定，在我 3部分描述。 <img src="/files/ftp_upload/1837/1837fig2.jpg" alt="图2" /> 图2：杨树（ 胡杨tremoloides） 杨树木材的木质纤维综合分析木片所描述的协议。 左上：矩阵多糖成分; FUC岩藻糖，RHA鼠李糖，阿拉伯糖阿拉伯糖，木糖木糖，人甘露糖，半乳糖半乳糖; GLC葡萄糖，肌醇内部标准。

<ol>
	<li>Albersheim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+analysis+of+sugars+in+plant+cell+wall+polysaccharides+by+gas-liquid+chromatography.">A method for the analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography.</a> Carbohydr. Res. 5, 340-340 (1967).</li><li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annu Rev Plant Biol. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+I:+Lignin.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part I: Lignin.</a> Journal of Visualized Experiments. , (2010).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterisation+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endotransglycosylases.">Discovery, characterisation and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endotransglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li><li>UPDEGRAF, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEMIMICRO+DETERMINATION+OF+CELLULOSE+IN+BIOLOGICAL+MATERIALS.">SEMIMICRO DETERMINATION OF CELLULOSE IN BIOLOGICAL MATERIALS.</a> Anal. Biochem. 32 (3), 420-420 (1969).</li></ol>

描述的方法，使木质纤维植物的生物量组成的快速定量评估。该方法允许这种材料，包括糖组成，即基质多糖的半纤维素的结晶纤维素含量组成的决心。每人的各种分析方法的吞吐量不同。使用这里描述的协议，20个样本可以处理矩阵的多糖成分和结晶纤维素含量30。由于数据的最佳原料作物的定量性质，品种或基因型，可用于生产生物燃料的适用性评估。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Trifluoroacetic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T6508</td>
<td>&nbsp;</td>
<tr>
<td>myo-Inositol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5125</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Borohydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>213462</td>
<td>&nbsp;</td>
<tr>
<td>Pyridine</td>
<td>&nbsp;</td>
<td>J.T. Baker</td>
<td>3348-01</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Anhydride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>320102</td>
<td>&nbsp;</td>
<tr>
<td>Spectromax Plus 384</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Plus384</td>
<td>&nbsp;</td>
<tr>
<td>GC-MS</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>7890A GC/5975C MSD</td>
<td>&nbsp;</td>
<tr>
<td>5.5mm Stainless Steel Balls</td>
<td>&nbsp;</td>
<td>Salem Ball Company</td>
<td>(N/A)</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate heat spreader</td>
<td>&nbsp;</td>
<td>Biocision</td>
<td>Coolsink 96F</td>
<td>&nbsp;</td>
<tr>
<td>Retsch Mill</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>TissueLyser II</td>
<td>&nbsp;</td>
<tr>
<td>Heating block</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>Dri-block DB-3D</td>
<td>&nbsp;</td>
<tr>
<td>Sample concentrator</td>
<td>&nbsp;</td>
<td>Techne</td>
<td>FSC400D</td>
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comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part ii: carbohydrates

可再生，碳中性的，和可持续发展的原材料行业和社会的需要，已成为面向21世纪的最紧迫的问题之一。这再度引发了在植物产品作为工业原料使用液体燃料的运输生产的兴趣<sup&gt; 2</sup&gt;和其他产品，如生物复合材料<sup&gt; 6</sup&gt;。植物生物量的仍然是这个星球上最大的未开发储量<sup&gt; 4</sup&gt;。它主要是由细胞组成的墙壁，能源丰富的聚合物包括纤维素，各种半纤维素，多酚木质素<sup&gt; 5</sup&gt;，因此有时被称为lignocellulosics。然而，植物细胞壁的演变顽抗退化，如墙壁广泛促进整个工厂的强度和结构完整性。尽管其必要的刚性，细胞壁是一个高度动态的实体，是代谢活跃，如许多植物的生长和分化的细胞活动中起着至关重要的作用<sup&gt; 5</sup&gt;。由于墙的各种功能，内有不同的植物种类和细胞类型的墙壁上一个巨大的结构在一个单一的植物多样性<sup&gt; 4</sup&gt;。因此，根据什么样的作物品种，作物品种，或植物组织是一个生物炼制，解聚在处理化学/酶过程和随后的各种糖发酵的液体生物燃料的步骤需要进行调整和优化。这一事实的基础需要一个彻底的植物生物质原料分析。在这里，我们描述了一个全面的分析方法，使lignocellulosics组成的决心，是适合一个高通量分析中（图1）。方法开始准备destarched细胞壁物质。造成lignocellulosics再分割，以确定其单糖组成的半纤维素和其他基质polysaccharides1，其结晶纤维素含量<sup&gt; 7</sup&gt;。木质纤维素生物质中的木质素成分分析的协议是在第I部讨论<sup&gt; 3</sup&gt;。

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Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

植物生物量是一个主要的碳中性的可再生资源，可用于生产生物燃料的使用。植物生物量主要由细胞壁，一个结构复杂的复合材料，被称为lignocellulosics。在这里，我们描述了协议的内容和墙派生碳水化合物组成的综合分析。

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

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Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

29.9K 次观看.  Michigan State University (MSU). 植物生物量是一个主要的碳中性的可再生资源，可用于生产生物燃料的使用。植物生物量主要由细胞壁，一个结构复杂的复合材料，被称为lignocellulosics。在这里，我们描述了协议的内容和墙派生碳水化合物组成的综合分析。

视频: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

牛痘病毒感染及病毒基因表达的时空分析：第1部分

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

科研

生物学

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

牛痘病毒感染及病毒基因表达的时空分析：第2部分

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

牛痘病毒感染及病毒基因表达的时空分析：第3部分

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

无标签原位成像在植物细胞壁的木质化

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...