Este trabajo fue financiado por una subvención de proyecto piloto de centro CREH auspiciada por NIEHS y por la Fundación Nacional de Ciencias naturales de China (Nº 31572626).

Todos los experimentos en animales fueron ejecutados en cumplimiento de todas las directrices pertinentes, regulaciones y agencias reguladoras. El protocolo se demostró fue realizado bajo la dirección y aprobación de la institucional Animal Care y el Comité uso (IACUC) de la Texas A & M Instituto de medicina genómica.
1. preparación de la solución
<ol><li>Diluir una solución salina equilibrada de Hank de x 10 a 1 x con doble destilada H2O y almacenar a 4 ° C.</li>
	<li>Preparar la solución de aislamiento mediante la adición de HEPES (10 mM), MgCl2 (1 mM) y glucosa (5 mM). Ajustar el pH a 7,4 y mantener en hielo.</li>
	<li>Preparar la solución de colagenasa de colagenasa tipo 4. Nota: Especialmente, la colagenasa tipo 4 contiene 160 U por mg de peso seco. 1 g de colagenasa tipo 4 es diluido en 5 mL doble destilada H2O con una concentración de 200 mg/mL. se agrega 2 mL de colagenasa diluida a la solución de aislamiento frío de hielo de 30 mL y 6 mL de solución de colagenasa se transfiere a un tubo de 50 mL para cada ratón.</li>
	<li>Preparar la solución de lavado agregando 1 mM CaCl2 a la solución de aislamiento.</li>
	<li>Preparar la solución de purificación con solución de diatrizoato de sodio polysucrose frío (1,1119 g/mL, 5 mL).</li>
	<li>Preparar 500 mL de medio de cultivo de islotes pancreáticos mediante la adición de L-glutamina (20 mM), penicilina (100 U/mL), estreptomicina (100 μg/mL) y suero bovino fetal (FBS) (10%) en Medio RPMI.</li>
	<li>Prepare la solución DCFH-DA disolviendo el polvo en agua destilada a 200 μm y almacenar a-20 ° C. Nota: El ROS solución de tinción es recién hecho diluyendo DCFH-DA solución a la concentración final de 5 μm en RPMI. Este reactivo debe ser protegido de la luz.</li>
</ol>2. quirúrgico preparación
<ol><li>Inyectar por vía intraperitoneal (I.P.) un ratón con avertin (2,5% avertin, 0,4 mL/20 g). Después de que el ratón está totalmente anestesiado y no responde a hind pie pizcas, eutanasia el ratón y mueve la campana biológica.</li>
	<li>Coloque el ratón eutanasia con el abdominal hacia arriba y rociar la piel con etanol al 70% para la esterilización.</li>
	<li>Abrir la cavidad abdominal con un 1 cm U-incisión en el abdomen superior y retraiga la piel en dirección rostral sobre el pecho para exponer la cavidad abdominal.</li>
</ol>3. eliminación y perfusión páncreas
<ol><li>Como se ilustra en la figura 1, encontrar la ubicación de la ampolla. Utilizar un par de pinzas curvas para apretar el duodeno cerca de la posición del esfínter de Oddi. Usar otro par de pinzas curvas para sujetar a la pared del duodeno para bloquear la vía biliar en el duodeno. Nota: La ampolla no se ha insertado en este paso. Este paso está destinado a bloquear la vía biliar en el duodeno.</li>
	<li>Vuelva a colocar el ratón con la cabeza proximal y los pies distales en relación con el investigador.</li>
	<li>El conducto biliar común y la inyecte lentamente 3 mL de solución de colagenasa con una jeringa de aguja y 5 mL de 30 G. Asegúrese de que el conducto biliar común y el páncreas se inflan después de la inyección.</li>
	<li>Retire el páncreas distendido con los fórceps curvado y tijeras finas para separar el colon descendente, intestino, estómago y bazo. Colocar el páncreas en un tubo de 50 mL que contiene 3 mL de solución de colagenasa y mantenerlo en hielo.</li>
</ol>4. páncreas digestión
<ol><li>Coloque cada tubo de 50 mL en un baño de agua de 37 ° C. Incube durante 15-20 minutos (el tiempo varía con la cepa y edad de los ratones).</li>
	<li>Vibrar el tubo utilizando un vortex.</li>
	<li>Piscina 4-5 páncreas digerida en un colador grande Lavabo estéril y utilizar una cuchara para que el páncreas desintegrarse completamente. Utilice una jeringa de aguja y 10 mL de 23 G con solución de lavado para pipetear con fuerza las células digeridas fuera de la pantalla. Recoger la solución de lavado en un tubo de 50 mL.</li>
	<li>Centrifugar los tubos a 97 x g a 4 ° C por 1 min y decantar el sobrenadante.</li>
	<li>Añadir 25 mL de solución de lavado para tubos y vuelva a suspender el tejido con un vórtex suave. Desactivación de los tejidos a 97 x g a 4 ° C por 1 min y decantar el sobrenadante. Repita 3 veces. Tenga cuidado de no desplazar los tejidos concentrados.</li>
</ol>5. purificación de islotes
<ol><li>Añadir 10 mL de solución de purificación a la pelotilla de tejido lavado y resuspender suavemente el contenido.</li>
	<li>Recubrimiento con cuidado 5 mL de solución de aislamiento en la solución de purificación. Tenga cuidado de mantener una interfaz líquida sostenida entre las dos soluciones.</li>
	<li>Centrifugar el tubo a 560 x g a 4 ° C durante 15 min con una aceleración lenta y sin freno. Introduzca la pipeta en la interfaz y recoger la capa de islotes en nuevos tubos de 50 mL.</li>
	<li>Lave los islotes como se describe en el paso 3.5.</li>
</ol>6. revestimiento y tratamiento de las células del islote
<ol><li>Resuspender los islotes en 10 mL de solución de cultivo de islotes, suavemente mezcle bien y coloque 100 μL/pocillo con una pipeta multicanal a una placa de 96 pocillos. Colocar aproximadamente 5 islotes por 96 bien.</li>
	<li>Colocar la placa en la incubadora de cultivo de tejidos para 12 h.</li>
	<li>Centrifugar (112 x g) la placa de 96 pocillos y retire el medio de cultivo.</li>
	<li>Diluir los productos químicos ambientales en el medio de cultivo de islotes. Añadir 100 μl de cada uno de los diluyentes químicos en pocillos separados de la placa. Resuspender los islotes en cada pocillo e incubar durante 12 h a 37 ° C (la concentración varía para los diferentes productos químicos ambientales).</li>
</ol>7. ROS coloración y a la medida
<ol><li>Después del tratamiento de islotes, lavado con PBS 1 x (200 μL para cada 96-well). Nota: La concentración de las sustancias químicas es como sigue: AFB1: 3 μm, atrazina: 100 μm, BaP: 10 μm, BPA: 100 μm, β-estrógeno, PCB126: 10 μm, nonilfenol y BPA: 100 μm.</li>
	<li>Uso de N-acetil cisteína (NAC) como antioxidante para saciar ROS. Añadir 5 mM de NAC a los diluyentes químicos inducida por ROS e incubar durante 12 h a 37 ° C.</li>
	<li>Centrifugar la placa de 96 pocillos x 112 g e incubar con solución de 5 μm de sonda fluorescente (H2-DCFH-DA disuelto en medios RPMI) a 37 ° C durante 60 minutos.</li>
	<li>Lavar las células de los islotes tratados 3 veces con PBS y medir la fluorescencia con un lector de microplacas a 488 nm. Nota: La acumulación de DCF en células se puede medir por un aumento en fluorescencia en 530 nm cuando la muestra es excitada a 488 nm.</li>
	<li>Realizar la insulina estimulada por glucosa secreción (GSIS) ensayo9 de xenobióticos inducen ROS tratados islas e islotes no tratadas para medir los efectos de xenobióticos sobre la función fisiológica de los islotes pancreáticos.</li>
</ol>

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Los autores declaran que no tienen intereses financieros que compiten.

La acumulación de pruebas sugiere que la exposición a químicos ambientales desempeña un papel importante en el desarrollo de T2DM. Inducida por xenobióticos ROS ha sido reconocido como un factor etiológico potencial contribuyendo al desarrollo de T2DM. Los seres humanos están expuestos a una amplia gama de productos químicos xenobióticos y hay una gran necesidad de técnicas de investigación novedosas para identificar efectivamente los toxicantes pancreáticos e investigar el mecanismo de la toxicidad específica de las células pancreáticas.
En este estudio, basado en los procedimientos publicados en14, hemos desarrollado un protocolo para aislar islotes pancreáticos de ratón y la pantalla de las células con productos químicos xenobióticos para inducida por el estrés oxidativo a las células pancreáticas. También usamos este procedimiento para investigar las respuestas tóxicas específicas pancreáticas inducidas por los compuestos xenobióticos. Para obtener el páncreas de ratón, preferimos utilizar avertin sobre otros anestesia ya que no altera los niveles de glucosa en sangre y no afecta a la vasculatura del páncreas15,16,17. Encontramos que el paso de centrifugación del gradiente de densidad es crítico para el aislamiento de los islotes pancreáticos de alta pureza. Debe tenerse cuidado para obtener la capa distintiva entre la fase de amortiguamiento polysucrose sodio y aislamiento para obtener una muestra de islotes puro, desprovisto de escombros y exocrino las células/tejidos. El método puede utilizarse para el análisis en vitro de efectos químicos ambientales en la fisiología del islote pancreático, como el ensayo GSIS (como se muestra en la figura 3). Los islotes deben ser cuidadosamente dispersos con pipeteo repetido en celdas separadas para número de uniformados de la célula en la placa de cultivo celular de 96. El método descrito aquí combina el aislamiento de la isla y un ensayo rápido para la generación de ROS y por lo tanto, es una pantalla inicial simple y efectiva para los posibles productos químicos dañan el páncreas.
Utilizamos la secreción de insulina en respuesta a la glucosa para confirmar la identidad de los islotes pancreáticos aislados. El procedimiento es altamente adaptable para el análisis de los cambios en los parámetros, incluida la secreción de insulina (figura 3) y la generación de campo, en respuesta a xenobióticos tratamientos18.

Aumentos en la prevalencia de T2DM se han convertido en una crisis de salud global en los últimos años plantea una grave amenaza a la salud pública1. Se han encontrado muchos factores causal relacionada con el desarrollo de T2DM, entre los que repite resultados sugieren que un punto convergente común para estos factores es la inducción de estrés oxidativo que conduce a la generación excesiva de ROS2 , 3.
Una amplia gama de productos químicos ambientales como PCBs, dioxinas y BaP se ha encontrado para inducir el estrés oxidativo, que puede perjudicar el funcionamiento de las células β pancreáticas y llevar a resistencia a la insulina y T2DM4. Aunque a nivel fisiológico de ROS desempeña un papel importante en las funciones celulares, la exposición a ROS que excede la capacidad del sistema antioxidante provoca el daño a las células/tejidos y conduce a enfermedades5. Las células β pancreáticas expresan un bajo nivel de enzimas antioxidantes y por lo tanto están un objetivo sensible de la mediada por estrés oxidativo6,7. La exposición crónica a altos niveles de ROS se ha demostrado para causar la disfunción de células pancreáticas inducida por estrés5 así como resistencia a la insulina en el hígado y tejido adiposo8.
El objetivo general de este proyecto es desarrollar un ensayo basado en células para productos químicos de la pantalla para sus potenciales diabetogenic basados en la inducción de ROS en las células pancreáticas. El páncreas carece de desintoxicación metabólica y es un blanco sensible de daño inducido por xenobióticos6,7. Por lo tanto, midiendo directamente los ROS generados en las células pancreáticas, este análisis deben proporcionar una aproximación directa a la lesión inducida por el producto químico en el páncreas. Para desarrollar este método, aislados islotes pancreático de ratón, había cultivado el islote aislado bajo condiciones de cultivo celular con productos químicos y utiliza la generación de ROS inducida por químicos como la lectura. Este procedimiento es simple y eficaz en la identificación de productos químicos inductores de ROS en el islote aislado; pueden desarrollarse para la investigación de los mecanismos de toxicidad que son específicos para el páncreas in vitro.

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposición a determinadas sustancias químicas ambientales en humanos y animales se ha encontrado para causar daño celular de las células β pancreáticas que conducirá al desarrollo de diabetes mellitus tipo 2 (T2DM). Aunque los mecanismos para el daño de la célula β inducida por químicos eran probablemente sea complejo y confuso, un hallazgo recurrente es que estos productos químicos inducen estrés oxidativo que conduce a la generación de especies reactivas excesiva del oxígeno (ROS) que inducen daño a la célula β. Para identificar potenciales químicos ambientales diabetogenic, se aislaron células de los islotes pancreáticos de ratones C57BL/6 y las células del islote cultivadas en placas de cultivo celular de 96 pozos; Luego, las células del islote fueron dosificadas con productos químicos y la generación de ROS se detectó por 2', 7'-Diclorofluoresceína (DCFH-DA) colorante fluorescente. Usando este método, encontró bisphenol A (BPA), Benzo [a] pireno (BaP) y los bifenilos policlorados (PCB), podría inducir a altos niveles de ROS, sugiriendo que potencialmente pueden inducir daño en las células del islote. Este método debe ser útil para la detección diabetogenic xenobióticos. Además, las células del islote culta también pueden ser adaptadas para el análisis en vitro de la toxicidad inducida por el producto químico en las células pancreáticas.

Una micrografía del islote aislado sano se muestra en la figura 2, en la cual islotes tienen una forma redonda u ovalada con relativamente uniforme de tamaño (aunque uniformidad de tamaño puede variar de una cepa a cepa). Investigamos a continuación las funciones del islote pancreático en un ensayo en vitro por aislar el islote y estimulando la secreción de insulina en los islotes de cultura. La figura 3 muestra el análisis típico del ensayo GSIS de C57BL/6 islotes de ratón aislado inducida por glucosa de 3,3 mM y 16,7 mM9.
El método de placa formato pantalla, hemos identificado varios xenobióticos que causó inducción significativa de ROS en las células del islote (figura 4). Nos trataron los islotes aislados con diferentes productos químicos durante 12 h y la atrazina, BPA, BaP y PCB126y β de estrógeno podría causar significativa inducción de ROS en los islotes aislados10,11,12 , 13.
Aunque no es igual a la inducción de diabetogenic ROS, existe amplia evidencia que indica el importante papel de las ROS en causar daño a las funciones fisiológicas de las células β pancreáticas, que conduce a T2DM8. Por lo tanto, el valor del trabajo es identificar estos ROS inductores químicos como la pantalla inicial. Además, los compuestos identificados pueden ser más analizados en vitro en el sistema de cultivo de células de islotes pancreáticos aislados descrito en el manuscrito. Hemos demostrado los efectos de los xenobióticos inducen ROS de glucosa estimulando la secreción de insulina de los islotes aislados, que brevemente diabetogenic datos para estos compuestos probados. Islotes pancreáticos pretratadas con PCB126 y BPA mostraron una disminución significativa en la secreción de insulina que el grupo control (figura 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57327/57327fig1v3.jpg"> Figura 1 : Procedimiento de perfusión del páncreas de ratón. El procedimiento consta de los siguientes pasos: (1) encontrar la ampolla y utilizar dos pares de pinzas curvadas para apretar la ampolla. (2) Inserte la aguja a través de la bilis común. (3) lentamente inyectar 3 mL de stock de solución de colagenasa en la jeringa de 5 mL. (4) recoge el páncreas dilatado a partir del duodeno. 
 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57327/57327fig2v3.jpg"> Figura 2 : Aislados islotes de ratón. Islotes pancreáticos aislados de ratón en medio de cultivo se muestran. Barra de escala de 500 μm.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57327/57327fig3v3.jpg"> Figura 3 : Secreción de insulina estimulada por glucosa de los islotes pancreáticos aislados. La secreción de insulina estimulada por glucosa dependiente de la dosis (1, 3.3 y 16,7 mM, trataron durante 15 min) se midió en islotes aislados (promedio 5 islotes por pozo), con una insulina de ratón ELISA kit. Barras de error indican desviaciones estándar. p < 0.05.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57327/57327fig4v3.jpg"> Figura 4 : Inducción de ROS por xenobióticos en islotes aislados de ratones C57BL/6. Islotes aislados (A) fueron tratados durante 12 h y teñidos con colorante de fluorescencia 2', 7'-Diclorofluoresceína (DCFH-DA) durante 1 hora. La fluorescencia se determinó mediante un lector de fluorescencia. (B) la química inducida por ROS se apagó por NAC (5 mM). (AFB1: 3 μm, atrazina: 100 μm, BaP: 10 μm, BPA: 100 μm, PCB126: 10 μm, nonilfenol, BPA: 100 μm y NAC: 5 mM). Barras de error indican desviaciones estándar. p < 0.05. <a href="/files/ftp_upload/57327/57327fig4v3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57327/57327fig5v3.jpg"> Figura 5 : Xenobióticos efectos sobre la secreción de insulina estimulada por glucosa de los islotes aislados. Islotes aislados se pretrata con PCB126, β-estrógeno y BPA (4 h) y se mantuvieron en medio de cultivo de islotes pancreáticos para 1 h. Se recogió el sobrenadante de los islotes para la medición de la insulina por una insulina de ratón disponibles comercialmente ELISA ensayo. Barras de error indican desviaciones estándar. p < 0.05.

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Aquí presentamos un protocolo para aislar células de islote pancreático de ratón para la detección de las inducciones de ROS por los xenobióticos con el fin de identificar los potenciales químicos xenobióticos diabetogenic.

Una proyección basadas en células de islotes pancreáticos murinos Diabetogenic ambiental productos químicos

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K vistas.  Hunan Agriculture University. Aquí presentamos un protocolo para aislar células de islote pancreático de ratón para la detección de las inducciones de ROS por los xenobióticos con el fin de identificar los potenciales químicos xenobióticos diabetogenic.

Video: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Páncreas murino aislamiento Islet

Investigación

Biología

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análisis transcriptómico de unicelular de células endocrinas pancreáticas de ratón

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Biología del Desarrollo

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

Análisis del consumo de oxígeno de los islotes pancreáticos no humanos

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Modelo de ratón de la Diabetes Tipo 2: El aislamiento de los islotes pancreáticos y células en Vivo 2-Photon imágenes de islotes intactos

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Medicina

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Un alto contenido<em&gt; In Vitro</em&gt; Islotes pancreáticos de células β plataforma de descubrimiento de replicación

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Un modelo de Ovo para probar compuestos de insulina-mimético

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Neurociencias

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal trasplante de islotes pancreáticos en modelo murino

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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 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.

Un protocolo simple de alta eficiencia para el aislamiento de islotes pancreáticos de ratones

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Análisis cuantitativo de la mitofagia en células β pancreáticas murinas

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Autor destacado: Evaluación del flujo de mitofagia en las células β pancreáticas utilizando enfoques complementarios efectivos y robustos 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Enfoques complementarios para interrogar el flujo de mitofagia en las células β pancreáticas

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Una proyección basadas en células de islotes pancreáticos murinos Diabetogenic ambiental productos químicos

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