这项工作是支持由美国国家科学基金会（授予没有。DBI 0519984）和美国国立卫生研究院（赠款。5R01GM078279）..

1）植物材料的制备<ol><li>原生质体可来自许多不同的植物物种和组织提供了正确的组合，消化酶的细胞壁是用 1 。之前进行全面的实验，一个小规模的消化的物质，是可取的，以评估protoplasting效率的组织，酶，等，估计％的阳性细胞的细胞分选。在这里，来自拟南芥幼苗根细胞类型特异性表达绿色荧光蛋白（GFP）是用来的原生质体。内皮和静态中心的标志是：P 可控硅 ：GFP和WOX5由 P的静态中心：绿色荧光 蛋白 2,3（图1） 。 </li><li>水培苗正根长成（0.22％W / V Murashige和Skoog基础培养基[适马]，1％的生长介质，通过它可以让一个尼龙过滤器（250微米网; NITEX）（Sigma公司;图2a）在phytatrays W / V蔗糖，0.05％W / V MES [2 - （N -吗啉）乙磺酸]，用KOH pH值5.7）。此外，植物可以种植尼龙过滤器（100微米网状）（图2b）在垂直位置的1％的琼脂平板上。 </li><li>使用上述过滤器的根的收获，不仅艾滋病，它也有利于秧苗容易额外的治疗，如果需要的话。该过滤器允许幼苗被转移大批新phytatrays或平板辅以利益的催化剂。例如，phytatray设置已用于特定细胞类型的转录响应分析在拟南芥中的氮治疗4。 </li><li>确保显微镜下，你的荧光标记是正确的表达（尤其是当使用的细胞类型特异性标志物的治疗方案，治疗本身可能影响）。在这种情况下，在荧光显微镜下（尼康，图1）幼苗检查。请注意，细胞类型特异性的荧光标记线下共聚焦显微镜，以确定哪些类型的细胞标记，并确定在表达变异的特点应当是最初。 </li></ol> 2）准备的protoplasting解决方案<ol><li>解散1.25％W / V纤维素（养乐多），0.3％瓦特/ V Macerozyme（养乐多），0.4 M D -甘露醇，20毫米的MES和20毫米氯化钾软化水（从1米的股票），并调整pH值至5.7 1米的Tris /盐酸pH值7.5。该解决方案将略有浑浊。 </li><li>热的解决方案，到55 ° C 10分钟（该解决方案将转明确），让冷却至室温，加入0.1％前W / V BSA（牛血清白蛋白），10毫米氯化钙 2，5毫米的β-巯基乙醇。 </li></ol> 3）收获的植物材料protoplasting <ol><li>根收获刮他们用手术刀的尼龙网眼，并存入一个包含protoplasting解决方案瓶。一般来说，10毫升protoplasting解决方案是每1500幼苗根系。 </li><li>在室温下摇一小时的烧瓶中，轻轻（75 RPM）。潜伏期较长时间可能会增加原生质体的产量，但也将增加protoplasting本身基因表达的影响。 </li><li>过滤器与一个40微米的细胞过滤器（BD猎鹰）原生质体的解决方案和除以15毫升以上锥形管（BD猎鹰）的解决方案。 </li><li>管旋转500克注意10分钟的摆动桶离心机离心速度，这将取决于原生质体使用的类型，它们的脆弱性和酶处理过程中产生的细胞碎片的数量。 </li><li>删除的大部分上清，重悬在余下的解决方案的原生质体，他们用显微镜检查（图3）。 </li><li>一个hemacytometer来估计原生质体的数量和密度。细胞密度将确定进样速度，每秒率的事件，因此，所需的总时间排序在流式细胞仪的细胞（见4.3节）。 </li><li> 3.7）要么直接进行流式细胞仪或清洗和重新悬浮在一个潜伏期的解决方案，如W5（154毫米125毫米，5毫米，5毫米的MES氯化钾氯化钙，氯化钠，用KOH调节pH值至5.7）或protoplasting解决方案，没有原生质体添加酶。在转录的变化看，特别是当它的重要性，以尽量减少样品暴露可能会影响基因表达的条件，如改变原生质体，因此，建议保持在protoplasting解决方案的原生质体，并继续保持英寸的缓冲区，尽快以流式细胞仪。 </li></ol> 4）荧光激活细胞原生质体排序<ol><li>打开和准备的细胞分选。在这里，FACSAria（BD）是使用。 </li><li>设置一个流流与100＆MU;米喷嘴和20 PSI鞘压力。 </li><li>细胞密度和进样速度可调节的基础上是否一个最好的收益或实现的速度最快需要特定的实验。我们已经成功地排序密度高达千万细胞/ ml。 </li><li>使用流式细胞仪的样本鼓动选项，以防止沉淀的原生质体。如果流式细胞仪堵塞是一个问题，有三种可能的故障排除步骤：1。执行样本行反吹。 2。稀贵的原生质体悬浮，减少密度。 3。清理重复过滤步骤（3.3），离心后和再悬浮原生质体的解决方案。 </li><li>准备的仪器来测量488纳米的激光前向散射（FSC），侧散射（SSC），并在三十〇分之五百三十零nm的绿色荧光蛋白和二十○分之六百一纳米红色自体荧光光谱（RSA）的激发后排放。这些在本质上是用来隔离GFP阳性的原生质体的唯一参数。在这里，电压设置如下：FSC - 60V，250V南南合作，GFP的350V和335V的RSA。需要注意的是最佳的电压设置为每一个流式细胞仪不同的，甚至会需要整个一生的细胞分选调整。 </li><li>开始成立前向散射对侧向散射dotplot。应用，使测量的事件情节为中心的的电压设置。 </li><li>下一步，创建一个绿色与红色荧光信号点图。应用的电压设置，使测量活动产生的情节集中在对角线人口在寻找一个野生型（非GFP）的原生质体悬浮。从GFP标记线的一个原生质体悬浮液会产生明确的人口，野生型样品中从未见过的绿色荧光事件。 </li><li>设置赔偿的限制，调整GFP和RSA之间的光谱重叠。适当的赔偿约束的设置将允许更好地从非- GFP的原生质体和碎片的GFP阳性的原生质体的分离。这里使用的限制如下：RSA，减17.91％的GFP。 </li><li>设置一个门，找出GFP阳性的事件，阴性对照的非- GFP的原生质体应该用来帮助确定门的边界（图4）。 </li><li>为了留下小碎片进行分析，实现向前散射截止。在FSC与SSC的情节可视化的GFP阳性的事件，以帮助确定截止的位置。在这里，截止设置为5000。请注意，流式细胞仪将算作碎片排序活动，并与高层次的碎片样本可能有不同％的GFP比预期的积极事态。这不一定是一个问题。然而，在更多的碎片样本，再排序将采取。 </li><li>根据实验和大量的细胞类型进行分析，流式细胞仪的精度模式设置以获得最佳的收益或最优排序细胞纯度。 </li><li> RNA的提取，准备适量的RNA提取缓冲液收集管（1.5 ml离心管）。有了这个设置，20000排序事件将产生一个总体积约100μL，350μL提取缓冲液（微试剂盒，QIAGEN公司的RNeasy）排序。排序完成后，细胞悬液，可在池顶部的混合样本。 </li><li>商店的样品或直接进行RNA提取。成功的微阵列分析已经预制排序从低至500事件中提取的RNA。在这里，我们使用的RNeasy微型提取试剂盒（QIAGEN公司），在WT的Ovation碧RNA扩增系统，FL -的Ovation基因生物素模块V2（NuGEN的）。 </li></ol>代表性的成果其中约1,500为期一周的老P 可控硅 phytatray：：GFP的幼苗产生约60,000原生质体（hemacytometer测量）。 2.6％65000流式细胞仪处理的事件被定义为GFP阳性和排序（图4b）。 八板约1,500为期四天的老P WOX5：GFP的苗（12000）取得了约3000万原生质体（hemacytometer测量）。 0.063％的1600万流式细胞仪处理的事件被定义为GFP阳性和排序（图4c）。 10,000排序事件通常用于RNA的提取及可产生20至140 ng总RNA（图5）。 <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="图1" /> 图1。在拟南芥根细胞类型特异性的绿色荧光蛋白标记线 。荧光显微镜图像与微分干涉对比（DIC）和GFP的过滤器上的Eclipse 90i显微镜（尼康）在MetaMorph软件（Molecular Devices公司）上运行。 DIC和GFP图像叠加可视化的目的。这种视觉实验中使用这两个标记线;a）芘SCR：：GFP和B）P WOX5：绿色荧光蛋白 。 <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="图2" /> 图2。在环境控制器，在phytatrays（a）或垂直定位的琼脂（二）水，植物生长的条件。幼苗生长。 <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="图3" /> 图3。植物原生质体的表达 GFP荧光显微镜图像与微分干涉对比（DIC）和GFP的过滤器上的Eclipse 90i显微镜（尼康）在MetaMorph软件（Molecular Devices公司）上运行。 DIC和GFP图像叠加可视化的目的。箭头表示爆裂细胞，细胞碎片和一个GFP阳性的原生质体。两个白线之间的距离是50微米。 <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="图4" /> 图4。 GFP阳性的原生质体的荧光激活细胞分选的野生型（A）P可控硅所得的原生质体：：GFP（B）或P WOX5：绿色荧光蛋白（C）标线进行了分析和排序与FACSAria（BD ）利用盖茨定义一个绿色dotplot（三十零分之五百三十○纳米; X轴）与红（二十○分之六百一纳米Y轴）的荧光。在每个小区10万事件。 GFP排序门范围内的事件突出的绿色。 <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="图5" /> 图5。代表10.000排序细胞RNA的提取细胞直接成RNA提取缓冲液（凯杰）排序，RNA纯化，并在2100生物分析仪（安捷伦）的浓度，纯度和完整性检查。三个重复标记线显示为。

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

原生质体，原则上可以来自多种植物组织，优化了有利条件，将大大提高RNA的质量和数量。 protoplasting解决方案和选修孵化缓冲会影响这方面的问题。 许多不同的荧光蛋白可以使用，这取决于所用流式细胞仪的功能，如GFP，RFP，YFP，CFP资格或他们的许多变种及衍生工具。标志物的表达不只是可以驱动细胞类型特异性启动子或增强子陷阱，如下所示，但也由你最喜欢的发...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

高分辨率，细胞类型特异性基因表达分析，大大提高了发育调控的理解和在任何多细胞生物体的环境刺激的反应原位杂交和报告基因的可视化，可以在有限的范围内为此目的使用，但对于高分辨率的定量。 RT - PCR或高通量转录全分析特定的细胞类型的RNA分离是必要的。细胞表达一种荧光蛋白标记的组织，在一个特定的细胞类型和随后的荧光激活细胞分选（FACS）的解离，使人们有可能足够数量的材料收集RNA提取，cDNA合成/扩增和基因芯片分析。 一个广泛的细胞类型特异性荧光记者行，是植物研究界。在这种情况下， 拟南芥根的两个标记线：P可控硅：GFP（内皮和静态中心） 和P WOX5：绿色荧光蛋白（静态中心） 。大量的幼苗（千）水培或平板和收获，以获得足够的根材料作进一步的分析。是通过细胞壁的酶消化细胞分解的植物材料。此过程使得使用高渗透压引起质壁分离和市售的纤维素酶，果胶酶和半纤维素酶释放到溶液中的原生质体。 GFP阳性细胞流式细胞仪使用可视化的绿色与红色的发射光谱由488 nm激光激发的原生质体。 GFP阳性的原生质体可以区分增加绿色比红色发射。原生质体是典型的排序直接到RNA的提取液和存储在稍后的时间进一步处理。 这种技术是显示简单和可行的。此外，它表明，它可以毫无困难地使用隔离足够数量的细胞转录组分析的，即使是非常稀少的细胞类型 （如静态的中心细胞）。最后，证明了拟南芥幼苗的生长设置，使无并发症治疗的植物细胞分选前（如生物或非生物的应激反应的细胞类型特异性分析）。讨论可能的补充使用植物原生质体的流式细胞仪。

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

一个隔离特定的细胞类型，从植物材料的方法是证明。这种技术采用特定类型的细胞，细胞分离和荧光激活细胞分选表达荧光蛋白的转基因标记线。此外，增长的设置是在这里建立，有利于治疗<em&gt;拟南芥</em&gt;细胞分选前的幼苗。

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 ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

Research

JoVE Journal

Biology

24.6K 次观看.  New York University. 一个隔离特定的细胞类型，从植物材料的方法是证明。这种技术采用特定类型的细胞，细胞分离和荧光激活细胞分选表达荧光蛋白的转基因标记线。此外，增长的设置是在这里建立，有利于治疗拟南芥细胞分选前的幼苗。

视频: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

科研

生物学

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...