这里显示的技术开发资金从能源部生物能源科学和国家科学基金会的赠款。

第1部分：成长和存储合适的植物材料<ol><li>根据我们的经验，年轻的黄化黄瓜幼苗下胚轴作为这些实验的细胞壁物质来源方便。黄瓜种子播种在湿纸在避光盒子，里面是在黑暗的内阁保持在设定房间温度恒定在26 ° C。精确的温度并不重要，为22 °和30 °之间的任何C应被罚款，但温度将决定如何快速苗达到一个适当的发展阶段。温暖的温度，更快的幼苗发展。我们通常使用时，他们已发展到约5厘米的长度，达到播种后3-4天的幼苗。重要的是，在黑暗中生长的幼苗，即使少量的光，影响幼苗发育率和细胞壁的特性，我们衡量这项技术。第3天，你可以窥视在框中，使用过滤光线暗淡的绿色，幼苗发育检查。 </li><li>幼苗迅速削减和小塑料盒，每盒100-150苗包装，并储存在-80 ° C在此温度下，他们仍然有用的几个星期。 </li></ol>第2部分：准备细胞壁样本<ol><li>从冰箱中冷冻切苗的小团体（8-10）被转移到一个绝缘容器-80冷冻块。 </li><li>角质层覆盖下胚轴是用金刚砂打磨。这是通过反复绘图之间的拇指和食指，这是涂厚浆的湿碳化硅粉末胚轴。这需要一些经验，知道使用正确的压力：压力太大，表皮层开始分解并撕裂;太少的压力，将不透的角质层。也有迅速开展工作，因为胚轴的冷冻解冻，弛缓性和难以管理。 </li><li>磨损胚轴浸泡在冰水中删除最坚持碳化硅和存储在冰水然后准备以类似的方式，而其余胚轴。 </li><li>的下胚轴切到所需长度，通常为1.2厘米，与一个新的单刃刀片，然后在玻片上保持一致。 </li><li>现在，我们需要拼合的墙，去除细胞SAP和促进夹紧。第二个载玻片放在8-10样本组顶部，形成一个三明治。 5分钟的载玻片上放置一个重量（400-500克）。为重，我们经常使用含有适量的水的烧杯中。 </li><li>可选步骤：根据实验，下胚轴可能在这一点上简短的热处理灭活。要做到这一点，我们结合玻片连同一副橡皮筋，把100毫升去离子水大会在室温下在一个容器中，并将其放置在在满功率的微波炉。随着我们的微波炉在50秒左右的水开始沸腾，我们停止微波15秒，煮沸后开始。热水迅速浇灭了，用冷水代替停止变性。您可能必须更改这些时间，您的微波可能比我们的不同。过度加热的样本变弱和容易折断。随着加热不足，是不是灭活内源性膨大和墙上的样品保留一些酸性pH值的响应。 </li></ol>第3部分：引伸设置<ol><li>墙上的样品，现在夹在一个恒定的力量引伸。这是一个定制的设备，树脂玻璃比色皿的一个组成控股皮基底墙样本和样品的顶端连接到一个可移动的钳位。可移动的夹具上安装，通过开放式位置传感器线圈通过一杆结束，LVDT或“线性可变差动变压器，电子检测到一个小的金属圆柱体的位置，或”核心“，即附着杆。杆的上端是一个可调节配重的杠杆。这杆施加向上的力的可调墙壁上的样本量。力调整，通过添加或删除校准的金属重物，杠杆的尽头。 </li><li>回到墙上的样品 - 胚轴样品的基础到底是用细镊子和根尖结束〜2-3毫米之间的可移动钳开放颌骨拾起。这钳是一个弹簧加载的鳄鱼钳的金属涂塑颌骨之间，以防止直接接触的金属表面与缓冲溶液或墙上的样品。根据我们的经验金属离子可以从钳位利奇和抑制墙上的能力，延长，所​​以我们保持金属覆盖。 </li><li>一方面控股的移动式钳大会，墙上的标本皮基底是目前机动铰链之间的两件树脂玻璃比色皿和试管片断汇集，并拧紧，从而锁定在试管底墙样品。 </li><li>移动式钳大会现在轻轻的释放，使全部力量的平衡力被转移到墙上的标本。我们经常使用的总配重20克墙样品准备从黄瓜下胚轴。其他墙体材料或实验可能需要不同的权重。 </li><li>试管充满缓冲液（200 UL）和试管的位置移动或向下调整螺丝，使移动式钳LVDT测量范围的低端。我们的LVDT连接的微机数据采集单元，所以我们通过计算机监控LVDT位置。我们有八个并行连接一台电脑，这使得我们可以同时运行8墙样本的LVDT的集会。电脑记录的每一次30秒的LVDT集会的位置</li></ol>第4部分：测量扩展响应酸的pH值或膨大 - 代表性的结果<ol><li> [实验]用于测量酸诱导的延伸，我们开始与本地墙样品（即不与热灭活）和中性缓冲添加到试管中。墙上的样品延长了几分钟，增加紧张，但扩展衰变后几分钟率较低。我们的电脑可以让我们监控样品长度的变化（也就是说，在可移动夹具的位置的变化，在实验开始后，），或者我们可以监测时间导数的位置，换句话说，延伸率。延伸率稳定与时间价值低。 </li><li> 〜20分钟后，中性缓冲被删除。我们用薄的金属管，从大规格注射针，迅速清除缓冲区，以最小的干扰墙壁或机械装配，连接真空泵。酸性缓冲液，然后补充说，有时1-2快速交流，以确保完整的交流酸性缓冲。然后我们坐视墙上的反应。通常情况下，我们可以在几分钟内检测的速度扩展。 60分钟后，我们通常有足够的信息来评估扩展响应，虽然在某些情况下测量可延伸至更长的时间。 </li><li> [第二个实验]膨大诱导细胞壁扩展的测量，我们与热灭活墙样品和酸性缓冲开始被添加到试管。在第一个实验中，细胞壁的延伸率逐渐稳定到低价值，因为缺乏功能膨大。 </li><li> 〜20分钟后，膨大的蛋白质添加到试管“扣球”与试管中的缓冲区10-20 UL膨大的解决方案。膨大迅速穿透细胞壁的样品，并在几分钟之内，我们看到，延伸率有所提高。这个扩展的反应可以遵循一个小时或更长时间。 </li></ol><img src="/files/ftp_upload/1263/fig1.png" alt="" width="600" height="469" /> 图1：准备细胞壁评估酸诱导或膨大诱导墙扩展的程序的大纲。

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+extension+of+isolated+cell+walls+from+growing+cucumber+hypocotyls.">Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls.</a> Planta. 177, 121-130 (1989).</li><li>Rayle, D. L., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acid+Growth+Theory+of+auxin-induced+cell+elongation+is+alive+and+well.">The Acid Growth Theory of auxin-induced cell elongation is alive and well.</a> Plant Physiol. 99, 1271-1274 (1992).</li><li>Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-induced+growth+of+Avena+coleoptiles+involves+two+mechanisms+with+different+pH+optima.">Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.</a> Plant Physiol. 99, 1556-1561 (1992).</li><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loosening+of+plant+cell+walls+by+expansins.">Loosening of plant cell walls by expansins.</a> Nature. 407, 321-326 (2000).</li><li>McQueen-Mason, S., Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+endogenous+proteins+that+induce+cell+wall+expansion+in+plants.">Two endogenous proteins that induce cell wall expansion in plants.</a> Plant Cell. 4, 1425-1433 (1992).</li></ol>

对于本演示中，我们用黄瓜下胚轴细胞壁，因为他们已经被证明是一个可靠的来源，很容易处理，并具有良好的灵敏度，响应墙样品。我们也有过良好的成功与其他树苗以及一些材料从超市，如年轻的菠菜叶和芹菜，墙壁。基本上，年轻，柔软，生长迅速的植物组织有可能使用这种技术可以很容易地测量，但坚韧，老，氧化或nongrowing的植物组织都不太可能因为细胞壁交联反应。有一些其他的东西...

measuring plant cell wall extension (creep) induced by acidic ph and by alpha-expansin

日益严重的植物细胞壁的特征表现为“酸的增长”，我们的意思，他们是在低pH值（&lt;5）1更具可扩展性着称的属性。植物激素生长素迅速刺激至少有一部分细胞伸长酸增长机制，2，3，幼茎和类似组织。生长素激活的H +泵的质膜，造成细胞壁溶液酸化。长城酸化激活expansins，这是内源性的细胞壁松蛋白 4，导致细胞壁产生细胞膨压在墙壁上创建的紧张局势。因此，细胞开始迅速放大。这“酸增长”的现象很容易测量隔离（非生物）细胞壁标本。细胞壁的能力进行酸诱导延伸不是单纯的细胞壁多糖（如果胶）的结构安排的结果，而是取决于活动 expansins 5。 Expansins没有任何已知的酶的活性，并检测膨大活动的唯一途径，是衡量其细胞壁扩展的感应。这个视频报告的细节膨大实验获得合适的墙体材料的来源和制备技术及酸诱导的延伸和膨大诱导墙样品准备种植黄瓜下胚轴的延伸。 要获得适当的细胞壁样品，黄瓜幼苗在黑暗中生长，胚轴削减和冻结在-80 ° C。冷冻胚轴磨损，夷为平地，然后在引伸计测量的一个特殊的比色皿的恒张力夹紧。为了测量酸诱导的延伸，墙壁最初在中性pH缓冲，导致在低活动expansins本地细胞壁的组成部分。当酸性pH值的缓冲液交换，ex​​pansins被激活和细胞壁迅速扩大。我们也证明了一个重组实验膨大活动。对于这部分，我们使用一个简短的热处理，在细胞壁样变性的原生expansins。这些灭活的细胞壁不延长，甚至在酸性的缓冲，但除了细胞壁expansins迅速恢复他们的能力，延长。

Watch this Scientific Journal Video about Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin at JoVE.com

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

我们证明一个恒定的力量引伸来衡量长期，延长植物细胞壁标本（蠕变）酸性缓冲区和膨大蛋白质引起的。

酸性α-膨大诱导测量植物细胞壁扩展（蠕变）

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

measuring-plant-cell-wall-extension-creep-induced-acidic-ph-alpha

Research

JoVE Journal

Biology

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

13.9K 次观看.  Penn State University. 我们证明一个恒定的力量引伸来衡量长期，延长植物细胞壁标本（蠕变）酸性缓冲区和膨大蛋白质引起的。

视频: Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

Title Cell Encapsulation by Droplets

通过飞沫标题细胞封装

科研

生物学

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

浇注和运行的一种蛋白质凝胶重用商​​业磁带

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

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 ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...