我们承认NIBIB，美国国立卫生研究院院内研究计划的研究支持。

样细胞大会和扭矩<ol><li> 2窗口组件放在一起，我们开始样品池建设。 放置到窗口的持有人窗口垫片。窗口的位置内衬窗口持有人（或它有时也被称为一个窗口，垫），使开放的眼线对面的持有人的键槽。在一个很小的角度，将持有人的窗口，窗口调整窗口持有人的关键标志线。在窗口两侧边缘轻轻下压。 </li><li>为了确保合适的密封和准确扭矩的样品池，我们轻轻大衣Spinkote润滑剂住房垫片与螺丝环。你的拇指和食指之间传播Spinkote非常少量。涂层薄，Spinkote看不见的薄膜螺纹环线程。同样，大衣住房垫片。擦去任何可见的润滑剂。 </li><li>样品电池组装，开始由一个窗口组件滑动的窗口，面向您进入细胞内的住房，调整住房键与键槽。住房的关键核心键槽对齐，并让它轻轻地落在窗口大会在细胞内的顶部。 </li><li>切勿使用任何工具，推入细胞住房的核心。这可能会导致永久性损坏泄漏造成的CP在实验过程中。打开第二个窗口组件，使窗口是面向核心，远离你，对准键槽与住房的关键和核心的顶部滑动下来。放置在顶部的住房垫片，然后，一个螺丝环，所以“出”字可见。用手指和对齐工具，手拧紧螺丝环。 如果CP不容易滑入住房每桶，首先对齐，然后放置在它上面的第二个窗口大会。窗口组件上的应用柔和的下调压力，在同一时间都滑入细胞住房。这样，我们避免直接CP上的迫切。 现在是一个好时机，看看里面的灰尘，绒毛和指纹细胞。您还会注意到牛顿环，表明仍然有CP和Windows之间的空气。这些后会消失申请扭矩。 </li><li>随着螺丝环比上涨和“出”看得见的话，将样品细胞内细胞扭矩夹头扭矩立场一路下滑。扭矩的立场处理申请恒压细胞固定到位。在一个连续的动作，拧紧螺丝环，以120-140磅之间。如果使用可调式千分尺扭矩扳手，将它设置为120-140 /磅之间，拧紧螺丝环，直到显示设置扭矩的扳手“点击”已达到。松开扭矩的立场处理，小心取出样品池，没有触摸的Windows。 </li></ol>装载和密封样品池<ol start="6"><li>沉降速度实验中，我们经常加载到每个200ul移液器使用的12毫米的核心部门的400ul的体积。顶部和“出”字可见的螺丝环加载孔样品池的位置。枪头中途滑动到左边的核心部门，通过加载孔，并慢慢地免除200ul参考的解决方案。重复一次，共400ul。使用相同的枪头和相同的技术，小心缓慢地填补400ul样品溶液的核心部门。 卷150-180 UL用于沉淀平衡实验，但应遵循同样的关怀和技术。 这是必须匹配参考和样品溶液的体积，尽可能密切。坚决配件到移液器吸头，慢慢地装载和分配解决方案，避免装载泡沫和使用这两种解决方案中的所有卷保持尽可能平等的援助相同的枪头。 </li><li>密封放置到每个装载确保它们之间的中心片和铝制外壳定位孔2个新的红色房屋插头垫圈紧贴的细胞。盖每一个住房插件。用手拧紧，一个小的平头螺丝刀。 </li></ol>在样品转子单元格对齐<ol start="8"><li>装载转子之前，确保样品细胞泄漏的情况下，在相反的立场，制衡是不超过0.5克，比样品池轻。开始余额为零。首先权衡的样品池，将在对面的CB的位置。皮减去样品电池重量的平衡，然后，权衡CB。从抗衡的附件孔中添加或减去的重量。确保从顶部重量不突出。 Reweigh，以确保.5克内抗衡的最终重量轻是不是相反的样品池。 </li><li>使用4位转子，放置到第4位的抗衡，是可见的白色箭头指向在T他方向的离心力。确保它是一路下来转子内。轻轻拧紧固定螺丝，发现抗衡对面的白色箭头上，直到抗衡坚决到位，但仍然能够对齐。插头面临的转子中心和“出”字可见的螺丝环，与房屋的位置平衡的样品池。 </li><li>从下面的转子，配合使用对齐工具抗衡上的标志线内的标志线（最接近的转子中心的标志）。拧紧螺钉将其锁定到位对齐抗衡。以同样的方式，与转子上的位置2内标志线对齐的样品细胞标志线。 </li></ol>欲了解更多信息，请<a href="https://sedfitsedphat.nibib.nih.gov/default.aspx">参阅</a> https://sedfitsedphat.nibib.nih.gov/default.aspx

<ol>
	<li>Brown, P. H., Balbo, A., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+protein-protein+interactions+by+sedimentation+velocity+analytical+ultracentrifugation.">Characterizing protein-protein interactions by sedimentation velocity analytical ultracentrifugation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.15-Unit 18.15 (2008).</li><li>Balbo, A., Brown, P. H., Braswell, E. H., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein-protein+interactions+by+equilibrium+sedimentation.">Measuring protein-protein interactions by equilibrium sedimentation.</a> Curr Protoc Immunol. Chapter 18, Unit 18.8-Unit 18.8 (2007).</li><li>. Home - Workshops [Internet] Available from: <a target="_blank" href="https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx">https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx</a></li></ol>

正确的电池组装，样品插入，并在转子单元格对齐方式是为高品质的AUC实验的关键。这才是真正的，特别是需要一个非常详细的分析，大分子沉积剖面上采集到的数据的实验，例如，在研究蛋白质相互作用，并确定跟踪总额。实验装置的质量通常是从AUC的研究结果的准确性和精度的限制。实用的设计和AUC的实验进一步执行的详细信息，请参阅参考文献1 - 3。工作坊覆盖的实际设置，理论和AUC的实验数据的分析，在我们的实验室正在举行每年两次在美国国立卫生研究院[4]。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>4-place Ti analytical rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361964</td>
<td>&nbsp;</td>
<tr>
<td>Torque stand assembly</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>361318</td>
<td>&nbsp;</td>
<tr>
<td>Cell alignment tool</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362340</td>
<td>&nbsp;</td>
<tr>
<td>Spinkote Lubricant</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306812</td>
<td>&nbsp;</td>
<tr>
<td>Counterbalance</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>360219</td>
<td>&nbsp;</td>
<tr>
<td>Cell Housing kit</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>334602</td>
<td>&nbsp;</td>
<tr>
<td>Window gasket</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327021</td>
<td>&nbsp;</td>
<tr>
<td>Window liner</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362329</td>
<td>&nbsp;</td>
<tr>
<td>Window, sapphire</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>307177</td>
<td>&nbsp;</td>
<tr>
<td>Window, quartz</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>301730</td>
<td>&nbsp;</td>
<tr>
<td>Window holder</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>305037</td>
<td>&nbsp;</td>
<tr>
<td>12mm double sector Epon centerpiece</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>306493</td>
<td>&nbsp;</td>
<tr>
<td>Cell plug gaskets</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>327022</td>
<td>&nbsp;</td>
<tr>
<td>Cell housing plug	</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>362327</td>
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assembly, loading, and alignment of an analytical ultracentrifuge sample cell

分析超速离心机（AUC）的是一个强大的生物物理的工具，使我们能够在高速离心大分子沉积剖面记录。当正确的规划和执行，AUC的沉降速度或沉淀平衡实验可以揭示蛋白质的大小和形状方面，样品纯度，沉降系数，齐聚国家和蛋白质相互作用很大。 然而，这种技术需要严格的技术注意水平。样品细胞举行一个扇面的中心片，夹在两个窗口组件之间。它们是密封的120-140 /磅左右的扭矩压力。参考缓冲器和样品装入的核心部门，然后密封后，细胞正是成为一个钛金属转子对齐光学检测系统，使每个核心部门通过扫描样品和参考缓冲器，而在相同的径向路径中线旋转速度高达60，000 rpm和非常高的真空下不仅是正确的样品池大会的关键，样本的细胞成分都非常昂贵，必须得到适当的照顾，以确保他们处于最佳工作状态，以避免在实验过程中的渗漏和破损。处理Windows小心，哪怕是一丁点的裂纹或划痕，可能会导致在离心机破损。的核心和Windows之间的联系必须尽可能紧;扭矩施加压力后，应该是可见的，即没有牛顿环。灰尘，棉绒，划痕和Windows或核心的所有妥协这种接触和油可以很容易导致泄漏的解决方案，从一个部门到另一个或泄漏的核心一起。不仅是珍贵的样品丢失，泄露的解决方案，在一个实验会造成不平衡，往往导致破碎的窗户和中心焦点在细胞内的压力。此外，插件垫圈和住房插头必须牢固到位，以避免解决方案的核心部门，通过在离心室的高真空的装载孔被拉。窗口衬里和衬垫必须免费断裂和裂缝可能导致运动破窗。 此影片将展示我们的样品电池组装程序，扭矩大，装载和转子对齐，以帮助减少元件损坏，泄漏的解决方案，在完美的AUC实验和破损。

Watch this Scientific Journal Video about Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell at JoVE.com

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

分析超速离心机（AUC）的样品池持有样品和参考缓冲器，并在实验过程中，暴露在高真空和转子速度高达60,000 RPM。此影片将展示装配，装载和对准这非常重要的组成部分的AUC实验所必需的细节一丝不苟。

大会，加载和分析超速离心机样品池对齐

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

assembly-loading-alignment-an-analytical-ultracentrifuge-sample

Research

JoVE Journal

Biology

22.4K 次观看.  Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science. 分析超速离心机（AUC）的样品池持有样品和参考缓冲器，并在实验过程中，暴露在高真空和转子速度高达60,000 RPM。此影片将展示装配，装载和对准这非常重要的组成部分的AUC实验所必需的细节一丝不苟。

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

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

大会，调整和使用蛋白成像Apertureless近场红外显微镜

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

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

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

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

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

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

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

对于样品制备与TEM分析低细胞数量的另一种方法

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

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

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

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

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...