Financiado por Sigma-Aldrich

1. Detección de Mycoplasma <ol><li> Sobrenadantes de cultivo celular se puede probar directamente, o la muestra se puede preparar para su uso en una fecha posterior. Para prepararse para su uso posterior a cabo 100 l de sobrenadante en un tubo estéril de amplificación y se incuba a 95 ° C durante 5 minutos. Una vez que se completa la muestra se puede almacenar a 2-8 º C durante un máximo de una semana. Justo antes de ejecutar el centrifugar brevemente muestra (5 segundos) para sedimentar los restos celulares. </li><li> Para preparar las muestras para PCR, determinar el volumen total de ADN Taq polimerasa Jumpstart / rehidratación búfer necesario para las reacciones. Vamos a preparar cinco reacciones total. Cinco reacciones requerirá 2.5μl de Taq y 114.5μl de tampón de rehidratación. Este contendrá un mínimo de una unidad de Taq por reacción y 22.5μl de tampón de rehidratación por reacción de la muestra y el control negativo más 24.5μl de rehidratación de amortiguación para el control positivo. 1 unidad de ADN polimerasa por reacción, debe añadirse a la cantidad adecuada de tampón de rehidratación. Esto va a variar con la Taq utilizado. </li><li> Coloque el volumen calculado de la ADN polimerasa Taq en un tubo de microcentrífuga limpio y seguir con el volumen calculado de tampón de rehidratación. La ADN polimerasa / rehidratación buffer se debe mezclar suavemente agitando el tubo. Esta mezcla no debe ser vortex. </li><li> Para preparar el control negativo y las muestras de uso de los tubos de reacción transparente en el kit. Los tubos de reacción en el kit ya contienen la nuclueotides, cebadores y el ADN de control interno. 23 l de mezcla de Jumpstart Taq ADN polimerasa buffer / rehidratación, como se prepara en los pasos anteriores, debe ser colocado en cada uno de los controles negativos y los tubos de muestra. Añadir 2 l de agua libre de ADN con el control negativo y añadir 2 l de la muestra a cada uno de los tubos de muestra y la etiqueta. Mezclar el contenido de revoloteo de los tubos. Contenido no debe ser vortex. </li><li> Para preparar el control positivo utilizar los tubos de reacción de color rosa en el kit. Los tubos de reacción en el kit ya contienen la nuclueotides, cebadores y el ADN de control interno. Añadir 25 l de mezcla de Jumpstart Taq ADN polimerasa buffer / rehidratación, como se prepara en los pasos anteriores, para los tubos de reacción y la etiqueta. Mezclar el contenido de revoloteo de los tubos. Contenido no debe ser vortex. Incubar el control negativo, control positivo y los tubos de la muestra a temperatura ambiente durante 5 minutos. </li><li> Para el PCR a tener lugar las muestras deben ser colocadas en un termociclador. Al utilizar JumpStart Taq un paso de activación no es necesaria. Coloque los tubos de reacción en el termociclador. Los ciclos deben establecerse de la siguiente manera: 94 ° C durante 30 segundos, 55 ° C durante 30 segundos y 72 ° C durante 40 segundos. Estos ciclos se ejecutará 40 veces. </li><li> Una vez que los ciclos de PCR se han completado las muestras deben ser enfriados hasta 4.8 ° C por dejarlas fuera del termociclador y colocarlos en hielo. Cuando se hayan enfriado las muestras que serán cargados en el gel de agarosa para electroforesis. De carga de gel y colorantes no son necesarios puesto que ya están presentes en los tubos de reacción. Directamente carga de 8 l para cada PCR en un carril separado. Detener la electroforesis después de la migración de 2,5 a 3,0 cm. </li></ol> 2. Resultados representante El control negativo debe mostrar una banda de aproximadamente 481 pb. Esto se puede ver en la columna de control negativo en la electroforesis en gel que se muestra al principio del vídeo. La ausencia de la banda de control negativo en 481 puntos básicos es un buen indicador de que la actividad de la polimerasa no se sufficienttaq utilizado no fue lo suficientemente sensible. Mycoplasma muestras positivas se muestran las bandas en el rango de 270 ± 8 pb. La banda será pesada para muestras muy contaminadas y débiles de las muestras ligeramente contaminados. Las variaciones en la intensidad se puede ver en las columnas muestra positiva en la electroforesis en gel que se muestra al principio del vídeo. Todas las muestras contienen un control interno negativo para demostrar que el PCR dio como se esperaba. Es normal que la ausencia del control interno en muestras muy contaminadas. Si no hay una banda en el rango positivo o una banda en el rango negativo en la muestra de esto es un buen indicador de que sus muestras están inhibidos. Si la muestra se inhibe los inhibidores de la PCR se pueden eliminar mediante la realización de una extracción de ADN. <img alt="Figura 1" src="/files/ftp_upload/3057/3057fig1.jpg" /> Las bandas de la figura 1 amplicón relevantes:. Resultados de la detección de micoplasmas por PCR se muestran. El carril 2 es el control negativo, que debe mostrar una banda de aproximadamente 481 pb. La ausencia de la banda de control negativo en 481 puntos básicos es un buen indicador de que el utilizado taq no era lo suficientemente sensible. Mycoplasma muestras positivas se muestran las bandas en el rango de 270 + 8 pb, como se muestra en los carriles de 3 a 6. La banda se heavy para muestras muy contaminadas (calle 6) y débil para las muestras ligeramente contaminados (calle 4). Todas las muestras contienen un control interno negativo (481 pb) para demostrar que el PCR dio como se esperaba. Es normal que la ausencia del control interno en muestras muy 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>

Los autores son empleados de Sigma-Aldrich que producen los reactivos y las herramientas utilizadas en este artículo.

Debido a la extrema sensibilidad de los kit, cuando se realiza correctamente el equipo debe dar resultados precisos que indica si la muestra tiene la contaminación por micoplasma. El kit ha sido optimizado para su uso con la polimerasa de ADN Taq JumpStart. Taqs se pueden usar otros en la preparación de las muestras, pero la banda de control interno en 481 pb, deben estar presentes para indicar la sensibilidad de la alternativa correcta taq. Es posible que las bandas positivas para exponer diferentes densidades en función del nivel de contaminación. Es posible observar la ausencia de la banda de control interno en muestras muy contaminadas. Precaución y utilizando la técnica adecuada en la preparación de las muestras para evitar la contaminación son fundamentales para la correcta. detección cuando se utiliza el kit de detección de Mycoplasma LookOut PCR.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> LookOut Mycoplasma PCR Detection Kit </td><td> Sigma-Aldrich </td><td> MP0035 </td><td> </td></tr><tr><td> JumpStart Taq ADN polimerasa </td><td> Sigma-Aldrich </td><td> D9307 </td><td> </td></tr><tr><td> Genómica GenElute sangre de ADN kit </td><td> Sigma-Aldrich </td><td> MP0030 </td><td> </td></tr><tr><td> Tubos Nunc amplificación </td><td> Sigma-Aldrich </td><td> T0447 </td><td> </td></tr></tbody></table>

optimized pcr-based detection of mycoplasma

El mantenimiento de líneas celulares libres de contaminación es esencial para la investigación celular. Entre las mayores preocupaciones de los contaminantes son la contaminación por micoplasma. A pesar de micoplasma no suelen matar a las células contaminadas, son difíciles de detectar y pueden causar una variedad de efectos sobre las células cultivadas, incluyendo la alteración del metabolismo, se redujo la proliferación y aberraciones cromosómicas. En resumen, la contaminación por micoplasma compromete el valor de las líneas celulares de proporcionar información rigurosa para la investigación en ciencias biológicas. Las fuentes de contaminación por micoplasma en el laboratorio son muy difíciles de controlar por completo. Como ciertas especies de micoplasmas se encuentran en la piel humana, pueden ser introducidos a través de una técnica aséptica pobres. Además, pueden provenir de los suplementos contaminados, tales como el suero bovino fetal, y lo más importante de otros cultivos de células contaminadas. Una vez micoplasmas contaminantes de una cultura, que pueden propagarse rápidamente a contaminar otras áreas del laboratorio. Estricto cumplimiento de buenas prácticas de laboratorio, tales como una buena técnica aséptica son fundamentales, y las pruebas de rutina para micoplasma es muy recomendable para el éxito del control de la contaminación por micoplasma. PCR basado en la detección de micoplasma se ha convertido en un método muy popular para el mantenimiento rutinario línea celular. Métodos basados ​​en PCR de detección son muy sensibles y pueden dar resultados rápidos, lo que permite a los investigadores para responder rápidamente a aislar y eliminar la contaminación una vez que se detecta en comparación con el tiempo necesario utilizar las técnicas microbiológicas. El kit de detección de Mycoplasma LookOut PCR es muy sensible, con un límite de detección de sólo dos genomas por microlitro. Tomando ventaja de la polimerasa de ADN altamente específicas JumpStart Taq y un primer diseño patentado, los falsos positivos se reducen considerablemente. El práctico formato de 8 tubos, tiras pre-recubiertos con dNTPs, primers y asociados ayuda a aumentar el rendimiento para satisfacer las necesidades de los clientes con grandes colecciones de líneas celulares. Dada la extrema sensibilidad del kit, el gran cuidado se debe tomar para prevenir la contaminación accidental de las muestras y reactivos. El protocolo de paso a paso se demuestra destaca las precauciones y prácticas necesarias para la detección de micoplasma fiable. También mostrar y discutir los resultados típicos y su interpretación. Nuestro objetivo es asegurar el éxito de los investigadores que utilizan el kit de detección 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

El kit de detección de Mycoplasma LookOut PCR utiliza la reacción en cadena de la polimerasa (PCR), que se ha establecido como el método de elección para la mayor sensibilidad en la detección de contaminación por Mycoplasma, Acholeplasma y Ureaplasma en cultivos celulares y cultivo celular otros derivados biológicos.

PCR optimizado basado en la detección 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 vistas.  Sigma-Aldrich. El kit de detección de Mycoplasma LookOut PCR utiliza la reacción en cadena de la polimerasa (PCR), que se ha establecido como el método de elección para la mayor sensibilidad en la detección de contaminación por Mycoplasma, Acholeplasma y Ureaplasma en cultivos celulares y cultivo celular otros derivados biológicos.

Video: 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.

Optimizado de gel de fibrina bolas de ensayo para el Estudio de la angiogénesis

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

Investigación

Biología

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.

La detección de ubiquitinación de proteínas

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.

La detección de metaloproteinasas de la matriz funcional por Zymography

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.

Un protocolo optimizado para cría Fopius arisanus, Un parasitoide de moscas de fruta tefrítidas

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).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y 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.

Protocolo optimizado para Retina Preparación Wholemount de la Imagen y la inmunohistoquímica

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.

Implantación de injertos vasculares Electrospun con estructura optimizada en un modelo de rata

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.

Optimizado el ensayo de rayado para In Vitro pruebas de migración de la célula con una cámara 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.

Detección simple de cilios primarios por inmunofluorescencia

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.

Detección y análisis automatizados de exocitosis

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