Name: effective isolation of functional islets from neonatal mouse pancreas
Uploaded: 2017-01-06T11:30:00
Description: Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.

el uso de animales sigue los procedimientos especificados en el protocolo M / 11/181 aprobada por la Vanderbilt Institucional Cuidado de Animales y el empleo de Gu. ratones CD1 o CBA / Bl6 fueron adquiridos de proveedores comerciales y se cruzaron en las instalaciones de animales de Vanderbilt para obtener ratones recién nacidos. 1. Preparación de los ratones, las soluciones concentradas y equipo <ol><li> Por la cruz del ratón: configurar una cruz ratón y registrar las fechas de tapona...

<ol>
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(50), (2011).</li><li>Zhao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galphao+represses+insulin+secretion+by+reducing+vesicular+docking+in+pancreatic+beta-cells.">Galphao represses insulin secretion by reducing vesicular docking in pancreatic beta-cells.</a> Diabetes. 59 (10), 2522-2529 (2010).</li><li>Planas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+for+the+human+pancreas+and+purified+islets+in+type+1+diabetes:+new+findings+at+clinical+onset+and+in+long-standing+diabetes.">Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.</a> Clin Exp Immunol. 159 (1), 23-44 (2010).</li><li>Tonne, J. 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A continuación, ofrecemos un protocolo paso a paso en el aislamiento de los islotes del páncreas que son demasiado pequeños para la perfusión convencional. Se espera para producir islotes listos para todos los estudios basados en los islotes como la purificación de las células beta, análisis de expresión génica, la maduración de las células beta de los islotes, la proliferación, las respuestas de estrés celular, la supervivencia celular, el metabolismo y mantenimiento funcional GSIS, etc. Esta ser?...

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies. 
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Collagenase&#160; Type IV</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:128px;">C5138</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:169px;">1X HBSS with Ca2+/Mg2+</td>
			<td style="width:253px;">Mediatech/Cellgro, Manassas, VA</td>
			<td>MT21020CV</td>
			<td style="width:219px;">If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively.&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:169px;">RPMI 1640 w/o glucose</td>
			<td style="width:253px;">Thermo Fisher Scientific/Life Technologies, Waltham, MA</td>
			<td>11879-020</td>
			<td style="width:219px;">Glucose needs to be added to specific levels to not interfere with seusequent islet usage.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Glucose</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>G-7021&#160;</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:169px;">polysucrose and sodium diatrizoate solution</td>
			<td style="width:253px;">Sigma Aldrich, St Louis, MO</td>
			<td>Histopaque-10771</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Carl-Zeiss, Oberkochen, Germany</td>
			<td>Stemi2000</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Stereoscope</td>
			<td style="width:253px;">Leica, Wetzlar, Germany</td>
			<td>Leica M165</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microcentrifuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5417C</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Centrfuge</td>
			<td style="width:253px;">Eppendorf, Hauppauge, NY</td>
			<td>Centrifuge 5810R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">15-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-666</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">50-ml centrfuge tubes&#160;</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>89039-658</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Precision balance</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>VWR-225AC</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Microfuge tubes</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>87003-294</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P1000</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123602</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Pipetman P20</td>
			<td style="width:253px;">Fisher Scientific,&#160; Waltham, MA</td>
			<td>F123600</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">100X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-088</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">60X15 millimeter dish</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>25384-168</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">12-well plates</td>
			<td style="width:253px;">VWR, Radnor, PA</td>
			<td>665-180</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">Scissor</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>14080-11</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:169px;">Tweezers</td>
			<td style="width:253px;">Fine Scientific Tools, Foster City, CA</td>
			<td>5708-5</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">CD1 mice</td>
			<td style="width:253px;">Charles River Laboratories, Wilminton, MA&#160;</td>
			<td>CD-1</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:169px;">C57BL/6J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>C57BL/6J</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">B6CBAF1/J</td>
			<td style="width:253px;">The Jackson laboratory, Farmington, CT&#160;</td>
			<td>B6CBAF1/J</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

effective isolation of functional islets from neonatal mouse pancreas

protocolos de islotes de aislamiento a base de perfusión de grandes páncreas de mamíferos están bien establecidos. Tales protocolos se llevan a cabo fácilmente en muchos laboratorios, debido al gran tamaño del conducto pancreático que permite la inyección de colagenasa listo y la perfusión tisular subsiguiente. En contraste, el aislamiento de los islotes de páncreas pequeña, como la de los ratones recién nacidos, es un reto porque la perfusión no se puede lograr fácilmente en la pequeña páncreas. Aquí se describe un procedimiento sencillo detallada que recupera un número sustancial de los islotes de ratones recién nacidos con asistencia visual. Recién diseccionado páncreas entero se digirió con 0,5 mg / ml de colagenasa IV disuelto en solución salina equilibrada de Hanks (HBSS) a 37 ° C, en tubos de microcentrífuga. Los tubos se intervenidos con regularidad para ayudar a la dispersión del tejido. Cuando la mayor parte del tejido se dispersó a pequeños grupos alrededor de 1 mm, los lisados ​​se lavaron de tres a cuatro veces con medios de cultivo con 10% de suero bovino fetal (FBS). racimos de islotes,desprovisto de tejidos acinares reconocibles, a continuación, se pueden recuperar bajo disección estereoscopio. Este método recupera 20 - 80 pequeño a islotes de gran tamaño por el páncreas de ratón recién nacido. Estos islotes son adecuados para los ensayos de aguas abajo más concebibles, incluyendo la secreción de insulina, la expresión génica, y la cultura. Un ejemplo de ensayo de secreción de insulina se presenta para validar el proceso de aislamiento. Los antecedentes genéticos y el grado de digestión son los factores más importantes que determinan el rendimiento. Se prefiere solución de colagenasa recién hecha con alta actividad, ya que ayuda en el aislamiento endocrino-exocrino. La presencia de cationes [calcio (Ca2 +) y magnesio (Mg 2+)] en todas las soluciones y suero fetal bovino en el lavado / medios de picking son necesarios para un buen rendimiento de islotes con integridad adecuada. Un microscopio de disección con un buen contraste y la ampliación también ayudará.

En condiciones óptimas, el método presentado puede rendimiento del 20 - 80 islotes de cada pequeño páncreas de ratón. Este número depende de los antecedentes genéticos, la edad de los ratones, y el tamaño de los islotes que debe recuperarse. Entre los de uso común, CD1 fuera criado y / 6J de pura raza C57BL producen menos islotes con un tamaño más pequeño que los híbridos entre CD1 y C57BL / 6 o comercial B6CBAF1 / J ratones hacen. Mano directa recogiendo generalmente dio un...

Watch this Scientific Journal Video about Effective Isolation of Functional Islets from Neonatal Mouse Pancreas at JoVE.com

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Se describe un protocolo en el presente documento para el aislamiento de islotes intactos de ratones neonatales. Páncreas se digirieron parcialmente con colagenasa, seguido de lavado y recolección a mano. 20 - 80 grupos de los islotes se pueden obtener por el páncreas de los ratones recién nacidos, que son adecuados para diversos estudios de los islotes.

El aislamiento efectivo de los islotes funcionales de páncreas de ratón neonatales

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

The overall goal of this procedure is to isolate intact islets of Langerhans from the neonatal mouse pancreas. This method can help answer the key questions in adding development and function. Such as insulin excretion, gene expression, para cell survival and functional maintenance.The main advantage of this method is that it's simple and straight forward while still allowing new researchers to isolate a substantial number of intact islet for down strain studies. Begin by spraying a neonate in the supine position with 70%ethanol. Then, use tweezers to lift the skin over the abdomen and cut the skin and muscle layers longitudinally along the midline from the genitals to the ribcage.Next, transfer all of the internal organs into a 100 centimeter dish and locate the pancreas, which is a white scattered organ with the ventral portion that clings to the duodenum and the dorsal portion that clings to the stomach and spleen. Add HBSS to dish to submerge the organs so that the pancreatic tissues no longer cling to the duodenum, stomach, or spleen. Then use a pair of tweezers to peel the pancreas away from surrounding tissues.Transfer the pancreas into a new 60 millimeter dish containing HBSS and use scissors to cut one to five pancreata into five millimeter pieces or smaller. When all of the tissue has been minced, transfer the pieces into one point five milliliter microcentrifuge tubes and add collagenase working solutions to the samples. Then incubate the tissue fragments at 37 degrees celsius, inverting the tubes two times every minute.After five minutes, tap the tube to monitor the digestion status of the pieces. Continue the digestion for another ten minutes, until the lysate is cloudy and most of the pancreata appear as fragmented cell clusters less than two millimeters in diameter. At the end of the digestion, pellet the lysate by microcentrifugation and use a p1000 pipette to carefully remove the supernatent, which should appear turbid but free of cell clusters.Next, add one millileter of fresh RPMI 16/40 complete medium to the pellet. Then gently tap to resuspend the fragmented lysate pieces and invert the tube ten times. After centrifuging, invert the resuspended lysate at least two more times as just demonstrated until the supernatent is clear.Then resuspend the final pellet in one milliliter of fresh complete medium and add two milliliters of polysucrose and sodium diatrazoate at a 1.077 g/ml density to a 15 milliliter conical tube on ice. Using a Pasteur pipette, carefully layer the lysate on top of the gradient solution without mixing the layers and separate the cells by centrifugation. Transfer the medium and the islet containing interphase layers into a new 15 milliliter tube and gently but thoroughly mix the islets with ten milliliters of fresh medium for centrifugation.After a second wash and fresh complete medium, resuspend the islet and duct containing pellet in four milliliters of fresh complete medium. Now seed the islets onto a 60 millimeter dish and add five milliliters of complete medium to the cells. Transfer the dish under a dissecting microscope and turn on the bright field illumination adjusting the light intensity to approximately 50%of the maximum power.At a 50x magnification, the islets will appear as slightly pink clusters and the acinar cells and dark, irregularly shaped clusters. Using a p20 pipette tip, harvest the individual islets, taking care to avoid the acinar clusters, dispensing the islet enriched fractions in a new 60 millimeter dish containing complete medium. When all of the islets have been picked from the original dish, harvest the islets from the collection dish into a new 60 millimeter dish containing four milliliters of fresh complete medium two more times, until only pure islets are present in the final collection dish.A proper digestion and isolation will yield islets with smooth outer surfaces. Insufficient collagenase digestion can result in large, easily visualized islets that can not be completely separated from the acinar tissues, reducing the overall islet yield even though larger islets are produced. In the case of over digestion, although acinar tissues can be completely separated from the islets, islet structure is prone to be compromised resulting in rough surface islets observed under the microscope.Neonatal islets isolated by this method display high basal insulin secretion levels with glucose stimulated insulin secretion profiles typical of immature islet beta cells, suggesting that this islet isolation procedure largely conserves the functional properties of neonatal islets. Once mastered, this technique can be finished within just two hours if performed properly. When attempting the procedure, it is important to remember to precentrifuge the collagenase in order to remove any insoluble debris and to calcium and magnesium supplemented washing buffer and to use pretreated washing buffer in order to stop the tissue dissociation quickly.For in this procedure, our experiment, such as glucose stimulated insulin secretion can be performed to answer additional questions such as insulin secretion dynamics in neonatal beta cells. Also, this technique pave ways for researchers in the field of beta cell biology to do additional essays, such as gene expression or other metabolic studies. After watching this video, you should have a good understanding of how to isolate intact islets from neonatal mouse pancreas for your down strain studies.Don't forget, working with collagenase powder will be extremely hazardous, so precautions such as wearing gloves and face mask should be always taken when using this material.

Research

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Rapid Isolation of Single Cells from Mouse and Human Teeth

Aislamiento rápido de células individuales de dientes de ratón y humanos

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Aislamiento de lisados de proteínas de células enteras de procesos faciales de ratón y células mesenquimas palatinas cultivadas para el análisis de fosfoproteínas

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...