Financiamiento proporcionado por el NIH (1 K08 DK080221-01) y los fondos de Vanderbilt de desarrollo institucional.

Parte 1: Modelo de reparación de lesiones Colitis <ol><li> Peso inicial se debe obtener para cada ratón antes de comenzar el tratamiento DSS. </li><li> Haga un 3% (w / v) de sulfato de dextrano sódico solución de sal en agua y filtrar con un filtro de acetato de 0,45 m de celulosa. </li><li> Reemplazar el agua potable en la jaula del ratón con la solución de DSS 3% durante cuatro días. Los ratones no deben tener acceso a cualquier otra fuente de agua (es decir, la exclusión consejos colocado en los sistemas de riego automático). </li><li> En el cuarto día, reemplazar la solución de DSS con agua por un período adicional de tres días, lo que permite una cierta recuperación del epitelio del colon. Los ratones se pesaron en el día cuatro con el fin de cuantificar las consecuencias sistémicas de la colitis. La pérdida de peso es común en lesiones graves. </li><li> El día 7, peso y sacrificio de los ratones. Los ratones pueden ser sacrificados por sobredosis por inhalación de isoflurano, o institucionalmente, IACUC métodos aprobados. </li></ol> Parte 2: La necropsia y recolección de Colón <ol><li> Exponer el lado ventral del ratón y las piernas seguro para garantizar el acceso sin obstáculos en el abdomen. Mojar el abdomen completamente con etanol al 70%. </li><li> Tome la línea media del abdomen con una pinza el tejido y se extienden hacia arriba lo que acampaban en la piel. Utilice tijeras de punta fina para incidir en el abdomen lo que expone el contenido abdominal. Extender la incisión a la punta de la apófisis xifoides en la línea media y luego se extenderá a lo largo de la cara inferior de los márgenes costal bilateral. </li><li> Identificar el intestino delgado, el ciego y colon. Diseccionar cuidadosamente / se burlan de los dos puntos del mesenterio rodea. Transecto del colon en la pelvis para liberar el colon distal y recto. Transecto de los dos puntos en el margen colonocecal para liberar el colon proximal. Se debe tener cuidado durante este proceso para no dañar el colon como la limpieza del colon será problemático si se produce una perforación. </li><li> Utilizando una aguja 20G de alimentación y una jeringa de 10 ml, intubar y limpiar el colon con helado de PBS hasta que el efluente está completamente libre de heces. </li><li> En este punto, si el análisis macroscópico con un microscopio de disección se desea, los dos puntos se puede fijar como &quot;salchichas&quot;. Si el análisis histológico sólo se desea que pase a la Parte 4 &quot;la fabricación de cilindros Suiza para el análisis histológico de colitis aguda&quot;. </li></ol> Parte 3: Tratamiento como &quot;salchichas&quot; para el análisis macroscópico de todo el colon <ol><li> Es importante mantener la orientación correcta del colon, por lo tanto, mantenga el extremo distal del colon más cercano al operador. Corte dos piezas de sutura no absorbible (SP116 1,5 seda trenzada métricas), uno de aproximadamente 1 pulgada de largo, los otros 2 pulgadas. </li><li> Utilice la pieza de 2 pulgadas de sutura para atar el extremo distal tan cerca de la margen posible sin dejar de mantener un buen sello. </li><li> Coloque una aguja 20G de alimentación que contiene 5 ml de tampón fosfato 10% de formalina en el extremo proximal del colon. Vagamente atar el pedazo restante de sutura inmediatamente proximal a la bombilla de la aguja de alimentación. Sujete firmemente el colon en el bulbo de la aguja de alimentación y de infundir en formol hasta que el colon se expanda. Apriete el nudo de la sutura que se retira la aguja de alimentación, lo que deja la infusión, colon ampliado como una &quot;salchicha&quot;. </li><li> Llenar un tubo cónico de 15 ml con tampón fosfato 10% de formol y el lugar de los dos puntos en el tubo. Fijar durante 24 horas. </li><li> Retirar la formalina y reemplazar con un 70% EtOH durante una 24 horas adicionales. (Los dos puntos se pueden almacenar en un 70% EtOH indefinidamente a temperatura ambiente.) </li><li> Quitar el colon desde el tubo cónico y cortar los hilos en cada extremo con un bisturí, con cuidado de no dañar el colon. Recuerde que el extremo distal tiene el mayor trozo de cuerda. Corte longitudinal de la porción distal de extremo proximal del colon de manera que forme una hoja larga. En este punto, el colon puede ser visto bajo un microscopio de disección. </li></ol> Parte 4: la fabricación de cilindros Suiza para el análisis histológico de colitis aguda <ol><li> Es importante mantener la orientación correcta del colon, por lo tanto, mantenga el extremo distal del colon más cercano al operador. </li><li> Medir la longitud del colon. Esta medida es otro indicador de la gravedad de la lesión. Colitis aumenta el edema y se acorta la longitud del colon en general. </li><li> Utilizando unas tijeras de punta fina, cortar longitudinalmente de distal a proximal final del colon. Use unas pinzas de punta fina para agarrar cualquiera de los bordes de la incisión y abrir lateralmente su forma de trabajo hacia distal-&gt; proximal, lo que muestra el colon como una lámina plana. </li><li> Rolling colon requiere un par de pinzas y una técnica a dos manos. Tome cualquiera de los bordes del margen distal con una pinza. Proceder a rodar de forma secuencial el colon mediante la rotación y la liberación de las pinzas de continuar con el borde proximal. Generando así una espiral con una tercera dimensión o &quot;rodillo suizo&quot;.Para mantener la forma enrollada, sujete firmemente el rollo con unas pinzas y transectos con una aguja 27G1 / 2. Asegurar la aguja al doblar la aguja a medida que sale del rollo. </li><li> Coloque el rollo en un casete de tejidos debidamente etiquetados y colocar en un frasco de tampón fosfato 10% de formalina a temperatura ambiente durante 24 horas para asegurar la fijación del tejido. </li><li> Retirar la formalina y reemplazar con un 70% EtOH durante una 24 horas adicionales. (Los dos puntos se pueden almacenar en un 70% EtOH indefinidamente a temperatura ambiente.) </li><li> Una vez que la muestra ha sido fijada, puede ser incluido en parafina, se cortaron y montaron en portaobjetos para su análisis histológico. </li></ol> Parte 5: Resultados de Representante El modelo DSS para la colitis aguda permite al investigador para obtener, corregir y analizar los dos puntos que la colitis aguda modelos. Cuando el brazo de gitano se corta y se monta, se debe formar una parte representativa de la totalidad del colon si se extiende adecuadamente (Figura 1). El rollo montado se puede teñir con H &amp; E con el fin de determinar el grado de daño al colon, de la parte distal (interior) el fin de la proximal (exterior) final (Figura 2). La inmunohistoquímica también se puede realizar en el brazo de gitano para identificar y cuantificar los infiltrados inflamatorios. Si el método de la salchicha se ha realizado correctamente, los dos puntos fijos se dilatan y toda la superficie de la mucosa puede ser fácilmente manipulado y se observa bajo el microscopio de disección (Figura 3). <a href="https://www.jove.com/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="Figura 1" border="0" /></a> Figura 1. Correctamente ejecutado &quot;brazo de gitano&quot;. (A) Los dos puntos se rueda desde la parte distal a proximal final, seccionado con una aguja y se asegura doblando el extremo de la aguja. Luego se coloca en una cinta de tejido para su fijación. (B) H &amp; E manchadas sección 5 micras de un brazo de gitano a partir de los dos puntos de un ratón tratado con DSS (d = colon distal p = colon proximal). <a href="https://www.jove.com/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="Figura 2" border="0" /></a> Figura 2. DSS dos puntos tratados muestran signos de colitis aguda. La inflamación y el daño cripta son evidentes en el colon DSS-tratados en comparación con un control del agua tratada. <a href="https://www.jove.com/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="Figura 3" border="0" /></a> Figura 3. Un ejemplo de una &quot;salchicha&quot;. La salchicha infundido con formol y se expandió por completo. Una ligera inclinación estará presente secundaria a la curvatura natural del colon. La salchicha abierta deben estar planos.

<ol>
	<li>Okayasu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Martinez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis.">Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis.</a> Gastroenterology. 131 (2), 579-588 (2006).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, G544-551 (1998).</li><li>Aharoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease.">Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.</a> J Pharmacol Exp Ther. 318 (1), 68-78 (2006).</li><li>Hahm, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease.">Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease.</a> Gut. 49 (2), 190-198 (2001).</li><li>Green, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+modulation+of+Escherichia+coli+O157:H7+adherence+to+the+colonic+mucosa.">Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.</a> Am J Physiol Gastrointest Liver Physiol. 287 (6), G1238-1246 (2004).</li><li>Dieleman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por Vanderbilt IACUC.

Este protocolo se puede modificar el modelo de lesión aguda, curación de las heridas, lesiones o procesos crónicos de colon. Modificación de lesiones agudas: Tratamiento DSS ad lib durante 4-6 días, seguido por el sacrificio inmediato Lesión-reparación de Modificación: Lesión con 4-6 días de tratamiento DSS seguido de un período de recuperación de 3-4 días y el sacrificio (como se describe en el pro...

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		<th>Catalogue Number</th>
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<tr>
<td>Dextran Sulfate Sodium Salt</td>
<td>&nbsp;</td>
<td>USB Corp</td>
<td>9011-18-1</td>
<td>mol wt 40,000-50,000</td>
</tr>
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<td>10% Buffered Formalin Phosphate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>SF100-4</td>
<td>&nbsp;</td>
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<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Phoenix Pharmaceuticals</td>
<td>NDC 57319-474-06</td>
<td>&nbsp;</td>
<tr>
<td>TISSUE PATH Macrosette Cassettes</td>
<td>&nbsp;</td>
<td>Fisher Healthcare</td>
<td>15182706</td>
<td>&nbsp;</td>
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<td>BD 10 ml Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309604</td>
<td>&nbsp;</td>
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<td>Ethyl Alcohol</td>
<td>&nbsp;</td>
<td>Pharmco-Aaper</td>
<td>E190</td>
<td>Dilute to 70% with distilled water</td>
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<td>20G Straight Feeding Needle</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20068-612</td>
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<td>27G1/2 PrecisionGlide Needle</td>
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<td>305109</td>
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<td>Dissecting Scissors Sharp/Blunt Tip</td>
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<td>VWR</td>
<td>82027-588</td>
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<td>Waugh Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-428</td>
<td>&nbsp;</td>
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<td>Non-absorbable Suture </td>
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<td>LOOK Surgical Specialties</td>
<td>SP116</td>
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<td>Whatman Blotting Paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>Cut as needed</td>
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<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-06731-2</td>
<td>&nbsp;</td>
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<td>Carbon fiber composites digital caliper</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-007-958</td>
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murine colitis modeling using dextran sulfate sodium (dss)

La colitis puede ocurrir por infecciones virales o bacterianas, ataque isquémico, o trastornos autoinmunes, colitis ulcerosa y en especial la variante de colon de la enfermedad de Crohn - Colitis de Crohn. Colitis aguda puede presentarse con dolor y distensión abdominal, mala absorción, diarrea, hematoquezia y moco en las heces. Estamos empezando a comprender las complejas interacciones entre el medio ambiente, la genética y la disfunción de la barrera epitelial en la enfermedad inflamatoria intestinal y los modelos animales de colitis han sido esenciales para avanzar en nuestra comprensión de esta enfermedad. Un modelo popular consiste en complementar el agua de bebida de los ratones con el sulfato de bajo peso molecular dextrano (DSS), que resulta en daño epitelial y una sólida respuesta inflamatoria en el colon de varios días<sup&gt; 1</sup&gt;. Las variaciones de este método se puede utilizar para modelar una lesión aguda, lesión aguda seguida de la reparación, y los repetidos ciclos de DSS intercalados con la recuperación de modelos de enfermedades inflamatorias crónicas<sup&gt; 2</sup&gt;. Después de un solo período de cuatro días de tratamiento de DSS del 3% en agua potable, los ratones muestran signos de colitis aguda, incluyendo la pérdida de peso, deposiciones con sangre y diarrea. Los ratones son sacrificados al concluir el curso del tratamiento y en dos puntos la necropsia disecados son procesados ​​y pueden ser &quot;suizo rodó&quot;<sup&gt; 3</sup&gt; Para permitir el análisis microscópico de todo el colon o infundido con formol como &quot;salchichas&quot; para permitir el análisis macroscópico. El tejido se incluyeron en parafina, seccionadas y teñidas para la revisión histológica.

Watch this Scientific Journal Video about Murine Colitis Modeling using Dextran Sulfate Sodium DSS at JoVE.com

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

De sodio de sulfato de dextrano (DSS), administrada en el agua potable es un modelo murino estableció una lesión inflamatoria de la colitis aguda. Este protocolo describe el método para el tratamiento de DSS y la preparación de los tejidos.

Modelado de la colitis murino utilizando sulfato de dextrano sódico (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

murine-colitis-modeling-using-dextran-sulfate-sodium-dss

Research

JoVE Journal

Biology

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

49.6K vistas.  Vanderbilt University. De sodio de sulfato de dextrano (DSS), administrada en el agua potable es un modelo murino estableció una lesión inflamatoria de la colitis aguda. Este protocolo describe el método para el tratamiento de DSS y la preparación de los tejidos.

Video: Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

La predicción de la eficacia de la estrategia de reemplazo de la población Utilizando modelos matemáticos

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Investigación

Biología

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Etiquetado linaje de células de pez cebra con láser Uncagable fluoresceína Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

La investigación de la inflamación intestinal inducida por DSS en el modelo de la EII

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

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 manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

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

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

La evaluación de la resistencia murino Función Arterial Utilizando miografía Presión

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

En Vivo Modelado del Genoma Humano Morbid usando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Proyección de imagen de FITC-dextrano como reportero para exocitosis regulada

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

In situ Exploración de la megacariopoyesis murina mediante microscopía electrónica de transmisión

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Evaluación de la integridad de la barrera intestinal en ratones utilizando dextrano marcado con fluoresceína-isotiocianato

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Simulación y análisis de bicapas lipídicas mediante dinámica molecular

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

Autor destacado: Avance de la biofísica de la membrana celular: exploración de interacciones y desafíos a través de enfoques experimentales y computacionales 

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

Modelado realista de membranas utilizando mezclas lipídicas complejas en estudios de simulación

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...