Nos gustaría agradecer a Renee L. Pasker y Harpreet K. Brar para la asistencia técnica de expertos sobre los protocolos descritos en este trabajo. Además, nos gustaría agradecer la tutoría de Christopher B. Newgard en la Universidad de Duke y Alan D. Attie en la Universidad de Wisconsin-Madison, junto con el apoyo de los miembros de su laboratorio, que nos permitió el tiempo y el apoyo necesarios para optimizar la protocolos descritos. En particular, agradecemos a Hans Hohmeier, Danhong Lu, y Helena Winfield en el Laboratorio Newgard y María Rabaglia en el Laboratorio Attie para los debates y asesoramiento de producción. Este trabajo fue apoyado por el NIH subvención DK080845 y Diabetes Juvenil 594 Fundación de Investigación de subvención 17-2011-608 (a MEK)

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Los autores tienen nada que revelar.

Con el predominio de la diabetes proyectados para afectar el 7,7% de la población mundial, la obligación de aplicar técnicas de investigación novedosas es imprescindible tanto para comprender y tratar la diabetes 18. El actual aislamiento de islotes es un protocolo bien establecido utilizado para la experimentación in vitro y se ha presentado con anterioridad, con ligeras modificaciones 11,14,16. Aunque la secreción de insulina es una aplicación común aguas abajo de islotes aislado...

El mantenimiento estricto de euglucemia es imperativo para prevenir la morbilidad como la neuropatía, nefropatía y retinopatía, que son todas las características de la patología de la diabetes 1 no controlada de tipo 1 y 2. Disminución de la función de las células β y la masa, tanto en la diabetes tipo 1 y 2 perturban las concentraciones de glucosa en la sangre 2. Considerando que el tipo inmunomediada 1 diabetes resulta de una devastadora pérdida de β-células productoras de insulina, la secreción de insulina de las células β alterada y la señalización de la insulina periférica en la diabetes tipo 2 en conjunto promueven la hiperglucemia, la dislipidemia y aumento de la producción hepática de glucosa, lo que finalmente se traduce en tanto la pérdida de la capacidad de la masa de células β y secretora de insulina a partir de β-células individuales 3. La comprensión de los mecanismos de las células β subyacentes en la progresión de la diabetes tipo 1 y 2 con suerte, dará lugar a nuevas terapias para prevenir y tratar estas enfermedades. En ti vitromodelos de cultivo ncidencia, como el INS-1 y MIN6 inmortalizados líneas de células β, pueden ser herramientas útiles para la comprensión de las funciones específicas de las células β. Sin embargo, las interacciones entre los diferentes tipos de células dentro de la isleta pueden ellos mismos regular la función de las células β. Por ejemplo, la influencia paracrina de glucagón (liberado de α-células) y la somatostatina (liberado de las células δ) en el aumento y la disminución de la secreción de insulina, respectivamente, demuestra la importancia de la proximidad de células de células en la respuesta endocrina 4. Por otra parte, los cruces brecha entre β-células potencian la liberación de insulina 5. Además, si bien se ha avanzado en la generación de líneas de insulinoma que mejor replican la respuesta fisiológica de los islotes aislados en glucosa (por ejemplo, el INS-1 derivado de 832/13 y 832/3 líneas celulares), su capacidad de respuesta de la glucosa aún difiere de rata normal islotes 6,7. Por otra parte, la respuesta de estas líneas celulares de insulinoma clonalesa péptido-1 (GLP-1) agonistas de tipo glucagón puede diferir drásticamente el uno del otro, así como de los islotes normales 6. Por lo tanto, las líneas de células inmortalizadas pueden no representar el mejor modelo para ensayar agentes que influyen en la producción de cAMP. En contraste con las líneas celulares de insulinoma-derivado, el estudio de la función de células β únicamente en modelos animales enteros ofrece su propio conjunto de complicaciones. Uno de los mayores retos en el trabajo con el tejido endocrino es la medición de la concentración exacta de la hormona liberada. En concreto, el hígado juega un papel importante el metabolismo de la insulina, y el flujo de sangre páncreas va directamente al hígado. Por lo tanto, una medición de la insulina en plasma no puede representar con precisión las cantidades de insulina secretada por el páncreas en sí o el impacto de diferentes tratamientos sobre la tasa de secreción de insulina 8. Además, el metabolismo renal de glucagón puede limitar la fiabilidad de la producción de glucagón de las células de los islotes α-9. Por lo tanto, el aislamiento de islotes primarios de ratón para la experimentación in vitro proporciona una comprensión más precisa de cómo el islote está respondiendo a los estímulos específicos para complementar las mediciones realizadas in vivo. El presente protocolo para el aislamiento de islotes de ratón es un protocolo bien establecido utilizado por varios grupos (con ligeras modificaciones que pueden ayudar a aumentar el éxito) 10,11. Además, la determinación de la producción de cAMP permite una lectura directa de la capacidad de respuesta de la incretina de las células β. En conjunción con la medición de AMPc, contenido de proteína y la secreción de insulina también se puede cuantificar a partir de la misma preparación de muestras de cAMP, lo que ayuda a determinar si un defecto en la función de las células β se encuentra proximal o distal a AMPc 10. El contenido de cAMP final y aplicación secreción de insulina en este protocolo puede ser una herramienta muy poderosa para la comprensión de la influencia de los componentes farmacéuticos y alimenticios, entre otross, el cAMP y la secreción de insulina. Además de la estimulación de glucosa sola, otros compuestos pueden ser utilizados para medir los cambios en el AMPc y la secreción de insulina 10,11. Por último, aunque la insulina es la principal hormona que del ensayo de islotes aislados, otras hormonas, tales como glucagón y somatostatina, así como citoquinas, eicosanoides, y monofosfato de adenosina cíclico, puede también ser medido, ya sea mediante un ensayo de estimulación transitoria o por cuantificación de sus niveles en el medio de cultivo 12. Por último, aunque fuera del alcance de este manuscrito, aislamiento de islotes con el método de aislamiento de colagenasa descrito permite la preservación de los islotes de manera que muchas otras aplicaciones posteriores pueden llevarse a cabo, tales como el trasplante de islotes, el aislamiento de ARN para cuantitativa en tiempo real PCR o análisis de microarrays, proteínas aislamiento para el Western Blot, la incrustación de los islotes y de formación de imágenes de inmunofluorescencia, y la incorporación de [3H]-timidina como una medida de la replicación de células de islotescatiónico, algunos de los cuales se han descrito en artículos anteriores JoVE 13-16. En general, siguiendo el procedimiento de aislamiento de los islotes se describe en el protocolo puede proporcionar un investigador con información útil e importante para el desarrollo de terapias y promover el descubrimiento de medicamentos destinada a mejorar la función de las células β.

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

Glucemia no controlada es una característica de la diabetes mellitus y promueve morbilidades como la neuropatía, nefropatía, y retinopatía. Con la creciente prevalencia de la diabetes, tanto inmune tipo 1 y relacionada con la obesidad de tipo 2, los estudios dirigidos a la delimitación de la fisiopatología de la diabetes y los mecanismos terapéuticos son de importancia crítica. Los β-células de los islotes pancreáticos de Langerhans son responsables de la secreción de insulina apropiadamente en respuesta a concentraciones elevadas de glucosa en sangre. Además de la glucosa y otros nutrientes, las células β también son estimulados por hormonas específicas, denominado incretinas, que se secretan en el intestino en respuesta a una comida y actúan sobre los receptores de células β que aumentan la producción de monofosfato de adenosina cíclico intracelular ( AMPc). Disminución de la función de las células β, la masa y la capacidad de respuesta de la incretina son bien entendidos para contribuir a la fisiopatología de la diabetes tipo 2, y también se están cada vez más vinculados widiabetes tipo 1 ª. El presente ratón aislamiento de islotes y el protocolo de determinación de cAMP puede ser una herramienta para ayudar a delinear los mecanismos que promueven la progresión de la enfermedad y las intervenciones terapéuticas, en particular los que están mediadas por los receptores de la incretina o receptores que actúan a través de la modulación de la producción de cAMP intracelular relacionado. Si bien sólo se describirán las medidas de cAMP, el protocolo de aislamiento de islotes descrito crea una preparación limpia que permite también para muchas otras aplicaciones posteriores, incluyendo la glucosa estimula la secreción de insulina, [3 H]-timidina incorporación, la abundancia de proteínas, y la expresión del ARNm.

Para asegurar un alto rendimiento durante el aislamiento de los islotes, las técnicas quirúrgicas descritas en el protocolo deben ser seguidos de cerca. Aunque las técnicas que aquí se presentan se adaptarán a cada laboratorio, hay algunos pasos críticos que conduzcan a un aislamiento exitoso. Con el fin de hacer que el conducto biliar común de fácil acceso, se recomienda que los órganos ser desplazados hacia el lado derecho del ratón (Figura 1). Además, esto permitirá que el páncreas se in...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

El ensayo in vitro función de las células β usando islotes aislados de ratones de Langerhans es un componente importante en el estudio de la fisiopatología de la diabetes y la terapéutica. Mientras que muchas aplicaciones posteriores están disponibles, este protocolo se describe específicamente la medición de intracelular de adenosina monofosfato cíclico (cAMP) como un parámetro esencial para determinar la función de las células β.

Un método para ratón islotes pancreáticos Aislamiento y cAMP intracelular Determinación

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Research

JoVE Journal

Biology

64.9K vistas.  University of Wisconsin-Madison.  El ensayo in vitro función de las células β usando islotes aislados de ratones de Langerhans es un componente importante en el estudio de la fisiopatología de la diabetes y la terapéutica. Mientras que muchas aplicaciones posteriores están disponibles, este protocolo se describe específicamente la medición de intracelular de adenosina monofosfato cíclico (cAMP) como un parámetro esencial para determinar la función de las células β. 

Video: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Murine Pancreatic Islet Isolation

Páncreas murino aislamiento Islet

Investigación

Biología

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Aislamiento humanos islote pancreático: Parte I: La digestión y la acumulación de tejido pancreático

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

De páncreas humano de aislamiento Islet: Parte II: Purificación y Cultura de los islotes

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Asistida por ordenador a gran escala de visualización y cuantificación de los islotes pancreáticos, distribución de tamaño y arquitectura

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Aislamiento de ratón Islet Langerhans utilizando una combinación de colagenasa purificada y proteasa neutra

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Aislamiento y cultivo de ratón primarias células acinares pancreáticas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Infrarrojo Cercano (NIR) Luz aumenta la expresión de un marcador de la función mitocondrial en el ratón vestibular sensorial Epitelio

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.