，由AGRON组学项目（拨款＃LSHG-CT-2006-037704），欧盟委员会在的框架化计划"的第 6，细胞类型和发展阶段的具体分析在布坎南沃拉斯顿实验室的工作是支持的。工作的吉福德实验室中细胞类型的具体分析是支持的的BBSRC研究者补助金（BB/H019502/1）的。 的KC274和KC464 拟南芥的线条得到了从AAR韦伯（剑桥大学）。

1。原生质体生成<ol><li>收获叶材料和切成1毫米宽的条带（ 图1）用剃刀刀片。对于含有较多的比单叶到15叶的样品可以容易地在切割之前层叠。 </li><li>马上转移叶条到一个9厘米的圆形培养皿中含有20毫升溶液（400mM甘露醇，20mM的MES水合物，20 mM KCl中的10mM的CaCl 2，2毫摩尔MgCl 2，0.1％BSA和2.5mMβ-巯基乙醇， pH 5.7）中，包括原生质体的酶（1.2％纤维素酶R-10，0.6％纤维素RS和0.4％离析酶R-10）和RNase和转录抑制剂（1×ProtectRNA和10微克/毫升放线菌素D），轻轻分开叶条和真空渗透为2×5分钟。 </li><li>广场上的陪替氏培养皿轨道摇床设置到第90转每分钟，在室温下和原生质体为3-4小时。 <ol><li>在温育的叶条应彼此分开的，通过使用一个平镊子，在半小时的时间间隔。 </li></ol></li><li>放置一个70μm的细胞过滤网（Fisher Scientific）的在50毫升的试管中，并轻轻的原生质体溶液通过过滤。这将删除的叶条，将保留筛，同时使原生质体去。 </li><li>叶洗净条用4毫升溶液A，轻轻地通过相同的筛过滤。 </li><li>将原生质体转移到圆底管和离心处理，以使在300×g离心5分钟以沉淀的原生质体的原生质体溶液。 </li><li>轻轻抽吸作为不干扰的原生质体的情况下尽可能多的上清液。 </li><li>洗净的原生质体，用5毫升溶液A，在300×g离心5分钟离心。除去上清液</li><li>重悬的原生质体在适当体积的溶液A中 ，使用巴斯德吸管或一个截止P1000小费的。 </li></ol> 2。 FACS的叶片原生质体</p&gt; <ol><li>排序，前立即重新悬浮使用（非切）P1000笔尖避免聚集在一起的原生质体的原生质体。 </li><li>将原生质体的分类管通过50μmFILCON的过滤器（BD Biosciences公司），并适当稀释，以避免堵塞喷嘴的细胞分选仪。的原生质体进行排序使用100μm喷嘴在压力20 psi（鞘）的和21-21.5 psi（样品）的，频率为39.2千赫的事件发生率&lt;4000（这些设置在BD流入细胞分选仪是最优（BD Biosciences公司），对其他细胞分拣机的优化可能需要调整这些设置）。 </li><li> GFP阳性的原生质体用488nm的氩激光和绘图580/30的带通滤波器（橙色） - 530/40的带通滤波器（绿色）的输出来识别。 GFP阳性的原生质体将在高530/low的580人口，非GFP原生质体高580人口低530 /低580人口和死亡/死亡的原生质体和碎片相关特征研（ 图2）。 <ol><li>进行排序时，从该RNA将被提取的原生质体，原生质体应直接分拣到至少4倍体积的含有还原剂（ 如 β-巯基乙醇）的裂解缓冲液。此外，对单个样品的排序时间应限制在20-30分钟，并在任何一个时间内处理的和最多两至三个样本。 </li><li>用于可视化排序的原生质体，将原生质体应直接排序条件到至少8体积的溶液A和其后通过离心收集集中的原生质体。 </li></ol></li></ol> 3。代表性的成果此协议描述了一种用于获得用于荧光激活细胞分选（ 图1），这是用来从拟南芥叶原生质体的方法。的存在下的原生质体产生的核糖核酸酶和转录抑制剂防止细胞fROM安装转录反应。这确实有许多死亡和垂死的原生质体，它有一个显着的自体荧光引致的后果。进行排序时，用这种方法制备的原生质体，因此，它是非常普遍地看到在580纳米 - 530 nm荧光曲线用于排序的图形的几个不同的种群。这方面的例子，连同在该协议中使用的分配门，在图3中所示。 原生质体可以排序成8倍体积的溶液A和收集用于可视化验证的富集。 图2G示出了一个这样的例子，使用的的TP382行，在保卫细胞中表达的GFP。 使用这种方法，两个前和扩增后的产率的实施例，示于表1。得到的金额是足够的Ovation微微WTA系统（500 PG-50毫微克），因此小号扩增ubsequent由任定量PCR分析（ 图4），:下一代测序或微阵列。 <img alt="图1" src="/files/ftp_upload/4214/4214fig1.jpg" /> 图1为实验的整体工作流程。 1）收获GFP的表达拟南芥叶兴趣。 2）快速切入〜1毫米宽的长条，并立即转移到原生质体的解决方案和真空渗透。 3）孵育3-4小时。 4）轻轻滤液原生质体解决方案，通过70筛，转移到圆底离心管和球团原生质体。 5）选前立即，重悬原生质体转移到排序管通过50微米的过滤器。 6）排序GFP阳性和的GFP负可行的原生质体。 7）原生质体现在可以进行分析，无论是视觉上或使用，如定量PCR技术，新一代测序或芯片。 图2。KC464，KC274和TP382本研究中所用的线。 A）的横截面的KC464叶的GFP表达在表皮。 B）原生质体衍生从KC464叶。 C）的横截面的KC274叶的GFP表达在血管。 D）原生质体来自KC274叶。 E）TP382叶保卫细胞中的GFP表达。 F）原生质体衍生从TP382叶。 G）例通过流式细胞仪的GFP表达的复杂背景下的叶细胞，保卫细胞中GFP含有原生质体的高530/low 580人口的从TP382叶原生质体衍生的富集。 AF:共焦显微镜图像; G:荧光显微镜图像。图像内的比例尺是50微米。 <img alt="图3" src="/files/ftp_upload/4214/4214fig3.jpg" /><html> 图3。典型的的FACS收购点图的输出从580/30纳米（橙色） -四十零分之五百三十nm（绿色）带通滤波器（BD涌入细胞分选仪）。的排序是那些通常用于在排序的门。 A）的点图上校-4叶衍生的原生质体，抑制剂的情况下，在原生质体，示出一个单一的低530/low 580人口。 B）的点图（重量）衍生上校-4叶原生质体，原生质体的抑制剂的存在下，示出了在580荧光移位由于压力和着色抑制剂。 CE）点图从叶派生原生质体KC74 KC464和TP382线，分别，抑制剂的存在下，在原生质体，示出了GFP包含在高530/low 580种群的种群。的事件在GFP（高530/low 580）人口的百分比。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4214/4214fig3large.jpg" target="_blank">点击这里查看大图</a> 。 e_content的"Fo:保持together.within"页面="总是"&gt; <img alt="图4" src="/files/ftp_upload/4214/4214fig4.jpg" /> 图4代表，如在图3中所示的门控人口馏分中的GFP表达。两个生物复制得到的各馏分的类型，使用GFP特异的引物（ 表2），然后进行使用的的Ovation微微WTA系统和qPCR扩增。所给出的值是最高的（红色），最低（绿色）和平均相对值。在这种情况下，高580人口一分为二，低530/low 580和高530/low的580，一个单一的生物复制使用只有单一的价值。 ACT2（At3g18780）使用所有的样品作为标准。叶:全叶。未分类的:未排序的原生质体。 GFP1:580和高530/low Rest1:低530/low 580，GFP2的:高530/high 580和Rest2:低530/high 580，如在图3和插入描述。结果是验证ØF光学评估GFP单元格内容的的排序分数（ 例如， 图2G）。 <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/4214/4214fig4large.jpg" target="_blank">点击此处查看大图</a> 。 <table border="1"><tbody><tr><td> 样品 </td><td> 叶片数 </td><td> 细胞的数目 </td><td> RNA产量 （NG） </td><td> RNA产量 （放大;纳克） </td></tr><tr><td>全叶甲 整个叶片B </td><td> 1 1 </td><td> - - </td><td> 8001 10525 </td><td> 4623 *** 4572 *** </td></tr><tr><td>未分类的原生质体阿 未分类的原生质体乙</td><td> 5 5 </td><td> - - </td><td> 2433 2625 </td><td> 1152 *** 3321 *** </td></tr><tr><td> GFP1一个 Rest1一个</td><td> 15 </td><td> 6039（0.39％*） 55389（6.40％*） </td><td> - ** - ** </td><td> 861 489 </td></tr><tr><td> GFP1乙 Rest1乙</td><td> 15 </td><td> 10783（0.63％*） 57040（7.49％*） </td><td> - ** - ** </td><td> 537 420 </td></tr><tr><td> GFP2 Rest2 </td><td> 15 </td><td> 18337（1.49％*） 62405（73.49％*） </td><td> - ** - ** </td><td> 360 411 </td></tr></tbody></table> 表1。样品收集，使用流式细胞仪和定量PCR，以确定哪部分包含现场表达GFP的原生质体进行分析。还包括本表中的总RNA产量，吴前和扩增后，采用Ovation微微WTA系统（NuGen）的。所有的馏分GFP1，GFP2的，Rest1和Rest2如在图4中所示的插入件。 *百分比细胞的总细胞排序运行的是，给定的括号中旁边的馏分中的细胞数。这些百分比是不相关的每个样品彼此的FACS排序运行"休息"部分早于由于更大数量的休息细胞的绿色荧光蛋白组分。 * 50纳克作为输入用于扩增反应，为制造商的协议。 <table border="1"><tbody><tr><td> 入门 </td><td> 序列 </td></tr><tr><td> mGFP5-ER.fw </td><td> GCCTGAGGGATACGTGCAGGAGA </td></tr><tr><td> mGFP5-ER.rv </td><td> TGGCGGGTCTTGAAGTTGGCT </td></tr><tr><td> ACT2.fw </td><td> GCCATCCAAGCTGTTCTCTC </td></tr><tr><td> ACT2.rv </td><td> GCCATCCAAGCTGTTCTCTC </td></tr></tbody></table> 表2中。qPCR检测。

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没有利益冲突的声明。

我们已经介绍了一种方法，使拟南芥的植物表达GFP的叶片原生质体生成和随后的细胞分选，使用流式细胞仪的使用。此方法产生足够量的RNA扩增，因此，随后的分析可用于定量PCR或芯片。排序的馏分高度富集含GFP-细胞，如通过qPCR证明（ 图2）。 为了达到高收率的活原生质体，它是至关重要的快速工作的程序的初始步骤期间，直到叶子条已经与含抑制剂的原生质体溶液真空渗入。此外，当生成1毫米叶条带，这是非常重要的切片或切叶而不是&#39;印章&#39;或捣烂他们，因为这会导致过多的损害和因此使用较低的原生质体产率。 一个关键的区别的方法描述在这里和其他Publishe公司ð方法是使用的RNase抑制剂和转录抑制剂。大多数方法拟南芥根系发达，从原生质体可以更容易地生成。然而，当工作用树叶，除了叶肉，它是必要增加的原生质体生成时间。因此，重要的是要防止基因表达的变化，作为结果的原生质体生成治疗本身（数据未示出），包括这些抑制剂。然而，抑制剂的存在下，会导致无法装入一个转录反应，或者通过切换，因为这会导致损失的可塑性，用以对付的应力影响的基因开启或关闭，这是有可能使原生质体更容易受到原生质体的过程。 我们已经使用这里描述的方法成功地与一些表达GFP的拟南芥线。更重要的是，我们已经使用了这种方法与GFP表达GFP，无论是强烈核，或更弱在一个non-localized/diffuse胞浆的方式，是与这里所使用的KC464和KC274线的的情况下。这两种类型的行是兼容的方法和得到的高度富集的馏分表达GFP的原生质体（ 图2和3）。该方法可以很容易地适合于其它荧光基团的荧光团，虽然必须选择是显着不同的死/死亡的细胞，以保存的能力来选择活细胞的自发荧光的荧光。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>纤维素酶R-10 </td><td>梅尔福德</td><td> C8001 </td></tr><tr><td>纤维素酶RS </td><td>梅尔福德</td><td> C8003 </td></tr><tr><td>离析酶R-10 </td><td>梅尔福德</td><td> M8002 </td></tr><tr><td> BSA </td><td> Sigma-Aldrich公司</td><td> A3912 </td></tr><tr><td> ProtectRNA </td><td> Sigma-Aldrich公司</td><td> R7397 </td></tr><tr><td>放线菌素D </td><td> Sigma-Aldrich公司</td><td> A1410 </td></tr><tr><td> 70微米的细胞过滤器</td><td>费舍尔</td><td> FB35181 </td></tr><tr><td> 50微米FILCON杯式过滤器</td><td> BD Biosciences公司</td><td> 340630 </td></tr></tbody></table>

cell specific analysis of arabidopsis leaves using fluorescence activated cell sorting

叶原基开始后，生物量积累主要由控制在叶片1的细胞的增殖和扩展。然而， 拟南芥叶片是由许多不同的细胞类型和几种结构的一个复杂的器官。同时在不同的发展阶段，越来越多的叶所含的细胞，细胞的最远的从叶柄第一个停止膨胀，并接受衰老1。因此，不同的细胞内叶划分，延长或差分活跃，压力或死亡和/或刺激的子集，它们的细胞类型在任何一个时间。这使得人类基因组研究的叶具有挑战性的:例如，当从全叶，信号从遗传网络操作在不同的细胞反应区或细胞类型表达数据分析将被混淆，导致所产生的一个不准确的更新。 为了解决这个问题，S已经描述了许多研究的方法，使细胞特异性基因表达的研究。这些包括激光捕获显微切割（LCM）2或表达GFP的植物用于原生质体的产生和随后的荧光激活细胞分选术（FACS）3,4，最近描述INTACT系统的核沉淀5和免疫沉淀的多聚核糖体6。 FACS已成功地使用了一些研究，包括示出小区标识和从根端部的距离，有一个显着的效果的大量的基因3,7上的表达谱。 FACS的GFP线也被用来证明在根氮反应和侧根发育8，盐胁迫9生长素在根10中分布的细胞特异性转录调控和创建的的拟南芥茎端分生组织11的基因表达的地图。虽然FACS以前被用来排序拟南芥叶片原生质体培养的自体荧光12,13，到目前为止，使用流式细胞仪在叶片中表达绿色荧光蛋白基因株系已经非常有限。4。在下面的协议中，我们描述了一种方法用于获得拟南芥叶片原生质体，用FACS兼容的原生质体生成机制的影响，同时最大限度地减少。我们证明了使用KC464的拟南芥线，其中表达GFP的表皮14，KC274线，在血管组织14和的TP382 拟南芥线，其中表达GFP表达双GFP构建体链接到一个核定位信号的方法保卫细胞（数据未示出， 图2）。目前，我们正在用这种方法来研究在不同阶段的衰老的细胞类型特异性表达在开发过程中的压力，以及异质细胞群。

Watch this Scientific Journal Video about Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting at JoVE.com

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

的制造方法<em&gt;拟南芥</em&gt;叶片原生质体，用荧光激活细胞分选（FACS）是兼容的，允许的特定细胞群的研究。此方法是与任何兼容<em&gt;拟南芥</em线，表达绿色荧光蛋白在细胞的一个子集。

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 ...

cell-specific-analysis-arabidopsis-leaves-using-fluorescence

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

15.2K 次观看.  University of Warwick. 的制造方法拟南芥叶片原生质体，用荧光激活细胞分选（FACS）是兼容的，允许的特定细胞群的研究。此方法是与任何兼容拟南芥

视频: Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

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 ...

科研

生物学

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

异核技术的细胞类型特异性定位分析

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

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 ...

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 ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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 ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

细胞特异性分析

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