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

53.5K 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 ...

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 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 ...

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 ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Otimizado zero ensaio para In Vitro testes de migração celular com uma câmera óptica automatizada

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Detecção Simples de Cília Primária por Imunofluorescência

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Detecção e Análise Automatizada de Exocistose

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...