Financiado pela Sigma-Aldrich

1. Detecção de Mycoplasma <ol><li> Sobrenadantes de cultura de células pode ser testado diretamente ou se a amostra pode preparados para uso em uma data posterior. Para se preparar para uso posterior mL colocar 100 do sobrenadante em um tubo de amplificação estéril e incubar a 95 ° C por 5 minutos. Uma vez completada essa amostra pode ser armazenado a 2-8 ° C por até uma semana. Pouco antes de executar a centrifugação breve amostra (5 segundos) para pellet quaisquer detritos celulares. </li><li> Para preparar as amostras para PCR, determinar o volume total de Jumpstart DNA polimerase Taq tampão / reidratação necessária para as reações. Estaremos preparando cinco reações total. Cinco reações exigirá 2.5μl de Taq e 114.5μl de tampão de reidratação. Isto irá conter um mínimo de uma unidade de Taq por reação e 22.5μl de tampão de reidratação por reação de amostra e controle negativo acrescido 24.5μl de tampão de reidratação para o controle positivo. 1 unidade de DNA polimerase por reação deve ser adicionado ao volume adequado de tampão de reidratação. Isso vai variar com o Taq usado. </li><li> Coloque o volume calculado de Taq DNA polimerase em um tubo de microcentrífuga limpo e seguir com o volume calculado de tampão de reidratação. A DNA polimerase tampão / reidratação deve ser misturado delicadamente sacudindo o tubo. Essa mistura não deve ser agitadas. </li><li> Para preparar o controle negativo e amostras de usar os tubos de reacção transparente fornecido no kit. Os tubos de reação fornecido no kit já contêm a nuclueotides, primers e DNA controle interno. 23 mL de Jumpstart Taq DNA polimerase mix de buffer / reidratação, elaborado nas etapas anteriores, deve ser colocado em cada um dos controle negativo e tubos de amostra. Adicionar 2 l da DNA água livre para o controle negativo e adicionar 2 mL da amostra para cada um dos tubos de amostra e etiqueta. Misture o conteúdo sacudindo os tubos. Conteúdos não devem ser agitadas. </li><li> Para preparar o controlo positivo use os tubos de reacção rosa fornecido no kit. Os tubos de reação fornecido no kit já contêm a nuclueotides, primers e DNA controle interno. Adicionar 25 mL de Jumpstart Taq DNA polimerase mix de buffer / reidratação, elaborado nas etapas anteriores, para os tubos de reação e de etiqueta. Misture o conteúdo sacudindo os tubos. Conteúdos não devem ser agitadas. Incubar o controle negativo, controle positivo e tubos de amostras à temperatura ambiente por 5 minutos. </li><li> Para a PCR para ocorrer as amostras precisam ser colocados em um termociclador. Ao usar JumpStart Taq uma etapa de ativação não é necessária. Coloque os tubos de reação no termociclador. Os ciclos deve ser definido da seguinte forma: 94 ° C por 30 segundos, 55 ° C por 30 segundos e 72 ° C por 40 segundos. Estes ciclos serão executados 40 vezes. </li><li> Uma vez que os ciclos de PCR ter concluído as amostras devem ser resfriado a 4-8 ° C, removendo-os do termociclador e colocá-los no gelo. Quando as amostras terem arrefecido devem ser carregados em gel de agarose para eletroforese. Carregando gel e tintura não são necessários uma vez que já estão presentes nos tubos de reação. Diretamente carga de 8 mL para cada PCR em uma faixa separada. Parar a eletroforese após a migração de 2,5-3,0 cm. </li></ol> 2. Resultados representante O controle negativo deverá mostrar uma banda em aproximadamente 481 pb. Isto pode ser visto na coluna de controle negativo na eletroforese em gel que é mostrado no início do vídeo. A ausência da banda de controlo negativo em 481 pb é um bom indicador de que a atividade da polimerase não foi sufficienttaq utilizado não foi sensível o suficiente. Mycoplasma amostras positivas mostrarão bandas na faixa de 270 ± 8 pb. A banda vai ser pesado para amostras altamente contaminadas e fraco para amostras levemente contaminados. As variações na intensidade pode ser visto nas colunas amostra positiva no gel de eletroforese que é mostrado no início do vídeo. Todas as amostras contêm um controle interno negativo para demonstrar que a PCR ocorreu como esperado. É normal ver a ausência do controle interno em amostras altamente contaminadas. Se não houver uma banda na faixa positiva ou uma banda na faixa de negativo em sua amostra este é um bom indicador de que as amostras são inibidas. Se a amostra for inibido os inibidores da PCR pode ser removido através da realização de uma extração de DNA. <img alt="Figura 1" src="/files/ftp_upload/3057/3057fig1.jpg" /> . Figura 1 bandas Amplicon relevantes: Resultados de detecção de micoplasmas por PCR são mostrados. Faixa 2 é o controle negativo, que deve mostrar uma banda em aproximadamente 481 pb. A ausência da banda de controlo negativo em 481 pb é um bom indicador de que a usada taq não foi sensível o suficiente. Mycoplasma amostras positivas mostrarão bandas na faixa de 270 pb + 8 como mostrado em faixas 3-6. A banda será heavy para amostras altamente contaminadas (faixa 6) e fraca para as amostras levemente contaminados (faixa 4). Todas as amostras contêm um controle interno negativo (481 bp) para demonstrar que a PCR ocorreu como esperado. É normal ver a ausência do controle interno em amostras altamente contaminadas.

<ol>
	<li>Wirth, M., Hauser, H., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+Sensitivity+of+Polymerase+Chain+Reaction+(PCR)+in+Comparison+with+Other+Methods+for+the+Detection+of+Mycoplasma+Contamination+in+Cell+Lines.">Specificity and Sensitivity of Polymerase Chain Reaction (PCR) in Comparison with Other Methods for the Detection of Mycoplasma Contamination in Cell Lines.</a> Journal of Immunological Methods. 164, 91-100 (1993).</li><li>Kong, H., Volokhov, D. V., George, J., Ikonomi, P., Chandler, D., Anderson, C., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+Cell+Culture+Enrichment+for+Improving+the+Sensitivity+of+Mycoplasma+Detection+Methods+Based+on+Nucleic+Acid+Amplification+Technology+(NAT).">Amplification of Cell Culture Enrichment for Improving the Sensitivity of Mycoplasma Detection Methods Based on Nucleic Acid Amplification Technology (NAT).</a> Applied Microbiology and Biotechnology. 77, 223-232 (2007).</li><li>Volokhov, D. V., Graham, L. J., Borson, K. A., Chizhikov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+Testing+of+Cell+Substrates+and+Biologics:+Review+of+Alternative+Non-Microbiological+Techniques.">Mycoplasma Testing of Cell Substrates and Biologics: Review of Alternative Non-Microbiological Techniques.</a> Molecular and Cellular Probes. , (2011).</li></ol>

Os autores são funcionários da Sigma-Aldrich, que produziu os reagentes e as ferramentas usadas neste artigo.

Devido à extrema sensibilidade do kit, quando realizada corretamente o kit devem produzir resultados precisos indicando se ou não a sua amostra tem contaminação por micoplasma. O kit foi otimizado para uso com JumpStart Taq DNA polimerase. Taqs outros podem ser usados ​​quando preparar as amostras, mas a banda de controlo interno a 481 bp deve estar presente para indicar a sensibilidade adequada do taq alternativa. É possível para as bandas positivas para expor várias densidades, dependendo do nível de contaminação. É possível observar a ausência da banda de controlo interno em amostras contaminadas. Usando cautela e boa técnica ao preparar as amostras para evitar a contaminação são cruciais para a adequada. detecção ao usar o Kit de Detecção de Mycoplasma LookOut PCR.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> LookOut Mycoplasma Kit de Detecção da PCR </td><td> Sigma-Aldrich </td><td> MP0035 </td><td> </td></tr><tr><td> JumpStart Taq DNA polimerase </td><td> Sigma-Aldrich </td><td> D9307 </td><td> </td></tr><tr><td> GenElute Sangue kit DNA genômico </td><td> Sigma-Aldrich </td><td> MP0030 </td><td> </td></tr><tr><td> Nunc tubos de amplificação </td><td> Sigma-Aldrich </td><td> T0447 </td><td> </td></tr></tbody></table>

optimized pcr-based detection of mycoplasma

A manutenção de linhas livres de contaminação de células é essencial para a célula-base da pesquisa. Entre as maiores preocupações são a contaminação por micoplasma contaminante. Apesar de micoplasma geralmente não matar células contaminadas, elas são difíceis de detectar e pode causar uma variedade de efeitos sobre as células cultivadas, incluindo o metabolismo alterado, diminuiu a proliferação e aberrações cromossômicas. Em suma, a contaminação por micoplasma compromete o valor dessas linhas celulares em fornecer dados precisos para pesquisa de ciências biológicas. As fontes de contaminação por micoplasma em laboratório são muito difíceis de controlar completamente. Como certas espécies de micoplasmas são encontradas na pele humana, eles podem ser introduzidas através de uma técnica asséptica pobres. Além disso, eles podem vir de suplementos contaminados, tais como soro fetal bovino, e mais importante de outras culturas de células contaminadas. Uma vez por micoplasma contamina uma cultura, ela pode se espalhar rapidamente para contaminar outras áreas do laboratório. Estrita adesão às práticas de laboratório, tais como a boa técnica asséptica são a chave, e testes de rotina para micoplasma é altamente recomendado para o sucesso do controle de contaminação por micoplasma. PCR baseada em detecção de micoplasma tornou-se um método muito popular para manutenção de rotina de linha celular. Métodos baseados em PCR de detecção são muito sensíveis e pode fornecer resultados rápidos, o que permite aos pesquisadores responder rapidamente para isolar e eliminar a contaminação, uma vez que é detectada em comparação com o tempo necessário utilizando técnicas microbiológicas. O LookOut Mycoplasma Kit de Detecção de PCR é altamente sensível, com um limite de detecção de apenas 2 genomas por microlitro. Aproveitando-se da Polimerase DNA altamente específicas JumpStart Taq e um desenho de primers proprietário, falsos positivos são muito reduzidas. O formato de 8-tubo conveniente, tiras pré-revestida com dNTPs, primers e associado ajuda a aumentar o rendimento para satisfazer as necessidades dos clientes com maiores coleções de linhagens celulares. Dada a extrema sensibilidade do kit, muito cuidado deve ser tomado para evitar a contaminação acidental das amostras e reagentes. O protocolo passo a passo, demonstramos destaca as precauções e práticas necessárias para a detecção de micoplasma confiável. Nós também mostrar e discutir os resultados típicos e sua interpretação. Nosso objetivo é garantir o sucesso de pesquisadores utilizando o Kit de Detecção de Mycoplasma LookOut PCR.

Watch this Scientific Journal Video about Optimized PCR-based Detection of Mycoplasma at JoVE.com

Optimized PCR-based Detection of Mycoplasma

O LookOut Mycoplasma Kit de Detecção de PCR utiliza a reação em cadeia da polimerase (PCR), que é estabelecido como o método de escolha para maior sensibilidade na detecção de contaminação por Mycoplasma, Acholeplasma e Ureaplasma em culturas de células e cultura de células derivadas outros produtos biológicos.

Detecção otimizada baseados em PCR de Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

optimized-pcr-based-detection-of-mycoplasma

Research

JoVE Journal

Biology

53.6K visualizações.  Sigma-Aldrich. O LookOut Mycoplasma Kit de Detecção de PCR utiliza a reação em cadeia da polimerase (PCR), que é estabelecido como o método de escolha para maior sensibilidade na detecção de contaminação por Mycoplasma, Acholeplasma e Ureaplasma em culturas de células e cultura de células derivadas outros produtos biológicos.

Vídeo: Optimized PCR-based Detection of Mycoplasma

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Otimizado de fibrina Gel Assay Bead para o Estudo da angiogênese

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Detecção de Ubiquitinação Protein

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Detecção de Metaloproteinases da Matriz Funcional por zimografia

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Um protocolo Optimizado para Criação Fopius arisanus, Um parasitóide de Frutas Tephritid Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Detecção de histona Modificações nas folhas das plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

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.

Optimizado coloração e Proliferação Métodos de Modelagem para Monitoramento Divisão Celular usando Corantes Rastreamento celulares

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

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Detecção de formas modificadas da citosina Utilizando imunohistoquímica Sensível

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

Implantação de enxertos vasculares Electrospun com estrutura otimizada em um modelo do rato

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...