Agradecemos o apoio de pesquisa pelo Programa de Pesquisa Intramural de NIBIB, NIH.

Assembléia amostra de células e Torque <ol><li> Começamos a construção de células de amostra reunindo duas assembléias janela. Coloque uma junta janela para o titular janela. Posicione o forro janela (ou às vezes é chamado de uma almofada janela) dentro do porta-janela para que a abertura do forro fica em frente à chaveta do titular. Em um pequeno ângulo, coloque a janela no porta-alinhando a marca escriba na janela com o modo como chave do titular janela. Pressione suavemente nas bordas muito em ambos os lados da janela. </li><li> Para garantir a vedação adequada e torque precisa da célula de amostra, que leve casaco a junta do alojamento eo anel de rosca com lubrificante Spinkote. Espalhe uma quantidade muito pequena de Spinkote entre o polegar eo indicador. Revista as roscas anel parafuso com uma película fina e invisível de Spinkote. Da mesma forma casaco, a junta de habitação. Limpe qualquer lubrificante visível. </li><li> Iniciar a montagem de célula de amostra, deslizando um conjunto de janela, com a janela voltada para cima em direção a você, na carcaça de célula, alinhando a ranhura com a chave da habitação. Alinhe a ranhura central com a chave da habitação e deixou-o suavemente cair em cima da janela de montagem dentro da célula. </li><li> Nunca use qualquer tipo de ferramenta para empurrar a peça central na carcaça celular. Isso pode causar danos permanentes ao CP, resultando em fuga durante as experiências. Vire o conjunto segunda janela para que a janela está virada para o centro, longe de você, alinhar chaveta com chave de habitação e deslize-o em cima do centro. Coloque uma junta de habitação no topo, então, um anel de rosca para a palavra &quot;Out&quot; é visível. Usando seus dedos e uma ferramenta de alinhamento, mão apertar o anel do parafuso. Se a CP não deslizar facilmente para o barril de habitação, primeiro alinhe-o, então, coloque o conjunto da segunda janela em cima dela. Ao aplicar uma pressão descendente suave na montagem janela, deslize ambos à habitação celular ao mesmo tempo. Desta forma, evitamos pressionando diretamente sobre a CP. Agora é um bom momento para olhar para dentro da célula para o fiapo, poeira e impressões digitais. Você também vai notar Anéis de Newton que indicam que ainda há ar entre a CP e as janelas. Estes irão desaparecer após a aplicação de torque. </li><li> Com o anel e parafuso até a palavra &quot;out&quot; visíveis, coloque a célula de amostra todo o caminho dentro da pinça torque de células do stand torque. Segure no lugar de células através da aplicação de uma pressão constante sobre a alça ficar torque. Em um movimento contínuo, apertar o anel de rosca para entre 120-140 in-lbs. Se estiver usando uma chave de torque ajustável micrômetro, configurá-lo para entre 120-140 in / kg e apertar o anel chave de parafuso até &quot;cliques&quot; que indica o torque set foi atingido. Solte a alça de pé torque e remova cuidadosamente a célula de amostra, sem tocar no windows. </li></ol> Carga e Vedação da célula de amostra <ol start="6"><li> Para experimentos velocidade de sedimentação, que rotineiramente carregar um volume de 400ul em cada setor da central 12 milímetros com uma pipeta de 200ul. Posição da célula de amostra com os orifícios de carga em cima ea palavra &quot;out&quot; no anel parafuso visível. Deslize a meio caminho da pipeta ponta para baixo para o setor central ESQUERDA através dos orifícios de carga e, lentamente, dispensar 200ul de solução de referência. Repetir uma vez ao total de 400ul. Usando a ponta da pipeta e mesmo a mesma técnica, cuidadosa e lentamente preencher o setor central DIREITO com 400ul de solução de amostra. Volumes de 150-180 ul são usados ​​para experimentos de equilíbrio de sedimentação, mas o mesmo cuidado e técnica devem ser seguidas. É imperativo para combinar com os volumes de soluções de referência e amostra, tanto quanto possível. Montagem da ponta da pipeta firmemente na pipeta, lentamente carregamento e distribuição de soluções, evitando bolhas de carga e usando a ponta da pipeta mesma para ambas as soluções toda a ajuda na manutenção dos volumes tão iguais quanto possível. </li><li> Vedação da célula, colocando duas novas juntas plugue vermelho habitação confortavelmente em cada buraco carregamento ter certeza que eles estão posicionados entre a peça central ea carcaça de alumínio. Cubra cada um com um plugue de habitação. Aperte com uma chave de fenda pequena plana. </li></ol> Alinhamento amostra de célula em Rotor <ol start="8"><li> Antes de carregar o rotor, certifique-se o contrapeso não é mais do que 0,5 g mais leve que a célula de amostra na posição oposta no caso de vazamentos de célula de amostra. Comece com o saldo a zero. Primeira pesar a célula de amostra que será em posição oposta ao CB. Tare a balança para subtrair o peso da amostra de células, então, pesar o CB. Adicionar ou subtrair o peso do furo de fixação do contrapeso. Certifique-se de pesos não se projetam a partir do topo. Pesar novamente para garantir que o contrapeso final é 0,5 g dentro mais leve que a célula de amostra oposto. </li><li> Utilizando um rotor de 4 lugares, coloque o contrapeso para a posição 4 para que a seta branca é visível e apontando em tele direção da força centrífuga. Certifique-se que é todo o caminho dentro do rotor. Gentilmente apertar o parafuso de ajuste, encontrado no topo do contrapeso do outro lado da seta branca, até que o contrapeso está firmemente no lugar, mas ainda capaz de ser alinhado. Posição da célula de amostra equilibrada com a habitação plugs de frente para o centro do rotor e da palavra &quot;out&quot; no anel parafuso visível. </li><li> Olhando de debaixo do rotor, alinhe a marca escriba dentro (a marca mais próxima do centro do rotor) com a marca de escriba na contrabalançar usando uma ferramenta de alinhamento. Aperte o parafuso de ajuste para bloquear contrabalançar alinhados no lugar. Da mesma forma, alinhar a amostra marca escriba celular com a marca dentro de escriba na posição 2 sobre o rotor. </li></ol> Para mais informações, consulte <a href="https://sedfitsedphat.nibib.nih.gov/default.aspx">https://sedfitsedphat.nibib.nih.gov/default.aspx</a>

<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>

O conjunto de células adequada, a inserção da amostra, e alinhamento de células no rotor é fundamental para experimentos de alta qualidade AUC. Isso é verdade, em particular, para experimentos que requerem uma análise muito detalhada dos dados adquiridos nos perfis de sedimentação macromolecular, por exemplo, ao estudar as interações proteína e determinação montantes agregados de rastreio. Muitas vezes a qualidade da montagem experimental é limitante para a exatidão e precisão dos resultados do estudo AUC. Para detalhes sobre o design prático e posterior execução de experimentos AUC, ver Referências 1-3. Workshops cobre a configuração prática, teoria e análise de dados de experimentos AUC estão sendo realizadas duas vezes por ano em nosso laboratório no Instituto Nacional de Saúde [4].

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assembly, loading, and alignment of an analytical ultracentrifuge sample cell

A ultracentrífuga analítica (AUC) é uma ferramenta poderosa biofísicos que nos permite gravar perfis de sedimentação macromolecular durante a centrifugação de alta velocidade. Quando devidamente planejado e executado, uma velocidade de sedimentação AUC ou experiência de equilíbrio de sedimentação pode revelar muito sobre uma proteína em relação ao tamanho e forma, a pureza da amostra, coeficiente de sedimentação, afirma oligomerização e interações proteína-proteína. Esta técnica, entretanto, requer um nível de atenção rigorosa técnica. Células da amostra segurar um pedaço centro sectored imprensado entre dois conjuntos janela. Eles são selados com uma pressão de torque de cerca de 120-140 in / lbs. Tampão de referência e amostra são carregados para os setores central e, em seguida, após a selagem, as células são precisamente alinhados em um rotor de titânio para que os sistemas de detecção óptica digitalizar ambos amostra e tampão de referência na linha média mesmo caminho radial através de cada setor central enquanto roda em velocidades de até 60, 000 rpm e sob vácuo muito alto Não só é a montagem de células de amostra adequada crítica, componentes de célula de amostra são muito caros e devem ser devidamente tratados para garantir que eles estão em ótima condição de trabalho, a fim de evitar vazamentos e quebras durante as experiências. Lidar com janelas com cuidado, pois mesmo a menor rachadura ou arranhão pode levar à quebra na centrífuga. O contato entre central e janelas devem ser tão apertada quanto possível; anéis ou seja, sem Newton s deve ser visível após a pressão torque é aplicado. Poeira, fiapos arranhões e óleos sobre o Windows ou o centro todo compromisso esse contato e pode muito facilmente levar ao vazamento de soluções de um setor para outro ou vazamento para fora do centro todos juntos. Não são apenas amostras preciosas perdidas, vazamento de soluções durante um experimento irá causar um desequilíbrio de pressão na célula que muitas vezes leva a janelas quebradas e centerpieces. Além disso, juntas plug and plugs de habitação deve ser firmemente no lugar para evitar soluções que está sendo retirado do setor central através dos orifícios de carga pelo alto vácuo na câmara de centrifugação. Liners janela e juntas devem estar livres de quebras e rachaduras que poderiam causar o movimento, resultando em janelas quebradas. Este vídeo vai demonstrar os nossos procedimentos de montagem de célula de amostra, a carga de torque e alinhamento do rotor para ajudar a minimizar danos aos componentes, a solução de vazamentos e quebras durante o experimento AUC perfeito.

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

A ultracentrífuga analítica (AUC) célula de amostra detém amostra e tampão de referência e durante as experiências e é exposto a alto vácuo e velocidades de rotor até 60.000 rpm. Este vídeo vai demonstrar a atenção rigorosa ao pormenor necessário para o carregamento, montagem e alinhamento deste componente muito importante de um experimento AUC.

Montagem, de carga, e alinhamento de uma célula Analytical ultracentrífuga Amostra

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 visualizações.  Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science. A ultracentrífuga analítica (AUC) célula de amostra detém amostra e tampão de referência e durante as experiências e é exposto a alto vácuo e velocidades de rotor até 60.000 rpm. Este vídeo vai demonstrar a atenção rigorosa ao pormenor necessário para o carregamento, montagem e alinhamento deste componente muito importante de um experimento AUC.

Vídeo: 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.

Dissecção do Organizador e Explantes Pólo animal a partir de embriões Xenopus laevis e Montagem de um ensaio de Adesão Celular

Biologia

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.

In Vitro Assembléia Nuclear Usando Extractos Egg fracionado Xenopus

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.

Assembléia, Tuning e Uso de um microscópio de campo Apertureless Near Infrared for Imaging Protein

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.

Uma ferramenta analítica que quantifica Alterações morfologia celular de imagens tridimensionais de fluorescência

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.

As amostras de teste para otimizar STORM Super-Resolution Microscopy

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.

Preparação da amostra para microscopia da força atômica de virion único e imagem de fluorescência de super-resolução

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.

Geração de plasmídeo vectores que expressam proteínas marcadas-bandeira nos termos do regulamento de Alongamento Human Fator-1α Promotor Usando Assembleia Gibson

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.

Uma abordagem alternativa para preparação de amostras com baixo número de celular de Análise TEM

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.

Fita de estomas-casca: Um método melhorado para preparação de amostras de células de guarda

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.

Amostra criogênica carregando em um espectrômetro de ressonância magnética nuclear de ângulo mágico que preserva a viabilidade celular

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