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.

Tiernutzung nach den festgelegten Verfahren in Protokoll M / 11/181 genehmigt von der Vanderbilt Institutional Animal Care und Use Committee für Gu. CD1 oder CBA / Bl6 Mäuse wurden von kommerziellen Anbietern gekauft und in der Vanderbilt Tieranlage gekreuzt neugeborenen Mäusen erhalten. 1. Herstellung von Mäusen, Stock-Lösungen und Geräte <ol><li> Für die Maus Kreuz: eine Maus Kreuz und notieren Sie die Aufstecken Termine einrichten experimentelle Planung zu unterstützen. CD1-Mäuse geben in der Regel Geburt um Tag 19 nach der Paarung. Verwenden Sie CD1 oder CBA / Bl6 kreuzt CD1 oder CBA / Bl6 Neugeborenen bzw. zu erhalten. Kreuzungen zwischen den beiden Linien nutzen CD1 Weibchen und CBA / Bl6 Männer. </li><li> Für die Kollagenase Lager: 200 mg Kollagenase Typ IV mit einem hohen Präzisionswaage wiegen. Transfer in ein 50 ml Zentrifugenröhrchen. Man löst in 40 ml HBSS mit Ca 2+ / Mg 2+ (1,26 und 0,5 mM jeweils) , um eine 5 mg / ml Stammlösung herzustellen. <ol><li> Lassen Sie Kollagenasefür ~ 30 min unter leichtem Schütteln lösen. Aliquotieren und die Lösung in -20 ° C als Vorrat eingefroren halten, die für mindestens ein Jahr lang aktiv bleibt. </li></ol></li><li> Autoklaven auf mehrere Paare von Pinzetten, Scheren und P20-Tipps für die Präparation und Inselchen Picking sterilisieren. </li><li> Für die 1 M Glucose Lager: wiegen 9 g Glucose in einem 50 ml Zentrifugenröhrchen, 50 ml RPMI 1640-Medium, das Glukose zu lösen. Halten Sie in 4 ° C bis zur Verwendung, aber nicht mehr als 6 Monate. </li></ol> 2. Arbeitslösungen HINWEIS: Der Tag der Inselisolierung, bereiten Sie die folgenden Reagenzien. <ol><li> Für die Kollagenase Typ IV Arbeitslösung: Tauwetter und Kollagenase Lager auf Eis verdünnt. Dispense 0,15 ml Brühe in jedem 1,7 ml Mikrozentrifugenröhrchen. In 1,35 ml HBSS mit Ca 2+ / Mg 2+. Drehen Sie das Röhrchen 5 mal. Lassen Sie auf Eis für 15 min. <ol><li> Spin bei hoher Geschwindigkeit für 3 Minuten in einer Mikro. Überstand in ein neues Röhrchen. Lassen Sie auf Eis bis zum Gebrauch. Entsorgen Sie das Rohr mit unlöslichen Ablagerungen. </li></ol></li><li> Zur vollständigen RPMI 1640 Medium: 2,5 ml 1 M Glucose, 50 ml hitzeinaktiviertem FBS und 5 ml 100x Pen-Strep Lager in 500 ml RPMI 1640 Medien hinzuzufügen. Lassen Sie auf Eis bis zum Gebrauch. </li></ol> 3. Pancreas Isolierung und Verdauung <ol><li> Euthanize Neugeborenen mit Isofluran, gefolgt von Enthauptung nach anerkannten Protokollen. </li><li> Legen Sie die Maus mit dem Bauch nach oben zeigt. Spray mit 70% Ethanol für die Sterilisation. </li><li> Heben Sie die Bauchhaut mit einer Pinzette und schneiden Sie die Haut und Muskelschichten in Längsrichtung entlang der Mittellinie mit einer Schere aus dem Genitalbereich des Brustkorbs. Dies öffnet die Bauchhöhle alle inneren Organe zu belichten. </li><li> Übertragen Sie alle inneren Organe zu einer 100-cm-Schale mit einer Pinzette. Suchen Sie die Bauchspeicheldrüse, die ein Streu Orgel mit einem Rückenteil ist , das in den Magen und Milz und eine ventrale Teil klammert , die 11 bis Duodenum klammert. </li&gt; <li> In HBSS in die Schüssel, die Organe zu versenken. In Lösung klammern die Pankreasgewebe nicht mehr auf den Zwölffingerdarm, Magen oder Milz. Es ist ohne weiteres erkennbar unter einem Binokular wegen seiner typischen weißen Farbe. Verwenden einer Pinzette die Pankreasgewebe weg vom umgebenden Gewebe und an einem neuen 60-mm-Schale mit HBSS zu schälen. </li><li> Schneiden Sie jede Bauchspeicheldrüse mit einer Schere in Stücke kleiner als 5 mm über beliebigen Abmessungen. Transfer zu einem 1,5 ml Mikrozentrifugenröhrchen. Bis zu fünf Bauchspeicheldrüsen (jünger als P7) kann in einem Röhrchen verdaut werden. Für P8-P16 Mäuse, verwenden Sie eine Röhre pro Bauchspeicheldrüse. </li><li> In 0,5-1 ml Kollagenase Arbeitslösung in jedes Röhrchen (0,2 ml pro P1-P7 Bauchspeicheldrüse Verwenden Sie 0,5 ml für jede P8-P16 Bauchspeicheldrüse.). Lassen Sie das Rohr in einem 37 ° C Inkubator für bis zu 15 min. Drehen Sie das Rohr zwei Mal pro min. </li><li> Nach ca. 5 min, tippen Sie auf das Rohr die Verdauung Status des Pankreasgewebe zu überwachen. Weiter den Verdauungsprozess, bis die meisten der PankreasGewebe erscheint als fragmentierten Zellcluster, mit einem Durchmesser von &lt;2 mm. Das Lysat wird trüb erscheinen, die den normalen Azinuszelltumoren Autolyse zurückzuführen ist. </li></ol> 4. Lysate Waschen <ol><li> Drehen Sie das Lysat bei 500 × g für 10 s in einer Mikro. Entfernen Sie die überstehende Schicht mit einem P1,000 Pipetman. Der Überstand sollte erscheinen trübe, aber ohne Zellhaufen. verwenden aspiration nicht, die die Gewebefragmente an der Unterseite durch die Anwesenheit von etwas klebrig DNA schnell abzusaugen weg kann. </li><li> 1 ml RPMI 1640 Komplettmedium. Tippen Sie auf das Rohr sanft die fragmentierte Lysat wieder zu suspendieren. Kehren nach oben und unten 10-mal. Wiederholen Sie Schritt 4.1. </li><li> Wiederholen Sie Schritt 4.2 zwei weitere Male oder bis der Überstand klar erscheint. Re-suspend gewaschenen Pankreas Fragmente in 1 ml komplettem RPMI 1640-Medium. </li></ol> 5. Islet Isolation HINWEIS: Bei einer kleinen Anzahl von Bauchspeicheldrüsen, direkte Handlese, wie unten (5.1) kann für die Insel isol verwendet werdenation. Für große Anzahlen von Bauchspeicheldrüsen (&gt; 6), wobei das Verfahren in 5.2 skizziert ist bevorzugt. <ol><li> Direkt Handlese: <ol><li> Übertragen Sie Lysat aus Schritt 4.3 zu einer 60-mm-Schale. In 5 ml komplettem RPMI 1640-Medium. Bringen Sie unter einem Binokular. </li><li> Stellen Sie die Hellfeldbeleuchtung auf dem Mikroskop auf. Stellen Sie die Lichtintensität auf ~ 50% der maximalen Leistung. Unter dieser Bedingung erscheinen Inseln als leicht rosa Cluster, während Acinuszellen als dunkle unregelmäßig geformte Cluster (Abbildung 1A). Eine Vergrößerung von ~ 50X (die Leistung des Objektivs zu kombinieren und das Augenteil) wird empfohlen, die beste Art und Weise zu visualisieren Inseln und die Pipettenspitze Öffnung gleichzeitig für die Kommissionierung zu bieten. </li><li> Pick-up-Inseln mit einem P20-Pipettenspitze während acinar Cluster zu vermeiden. Dispense Inselchen angereicherten Fraktionen auf eine neue 60 - mm - Schale mit RPMI Komplettmedium (1B). </li><li> Wiederholen Sie die Hand-Picking-Prozess noch zweimal. Lassen Sie rein Inseln in eine neue 60mm - Schale mit 4 ml komplettem RPMI Medium (1C). </li></ol></li><li> Gradientenzentrifugation: <ol><li> Vorbereiten eines 15 ml Zentrifugenröhrchen, die mit 2 ml Polysaccharose und Natriumdiatrizoat in einer Dichte von 1,077 g / ml vorgespannt ist. Lassen Sie das Röhrchen auf Eis bis zum Gebrauch. </li><li> Übertragung der Suspension aus Schritt 4.3 auf der Oberseite des Polysaccharose und Natriumdiatrizoat-Lösung mit einer Pasteur-Pipette. Sie nicht die untere Schicht stören. Gewebe von bis zu 10 neu geboren oder 2 P17 Bauchspeicheldrüsen können in jede 15 ml-Röhrchen geladen werden. </li><li> Drehe das Röhrchen bei ~ 600 xg in einem Schwenkrotor für 15 min bei 4 ° C. Verwenden Bremse-Aus-Einstellungen. </li><li> Nach Zentrifugation übertragen die Medien und Zwischenphasenschichten (man beachte, dass unter idealen Betriebsbedingungen werden die Inseln in der Zwischenphase zwischen Polysaccharose und Kulturmedien nach dem Zentrifugieren befinden) in ein neues 15 ml-Röhrchen. In 10 ml Medien mischen vorsichtig , aber gründlich. </li><li> Spin down die Insel angereicherte Fraktionbei 300 xg für 5 min. Entfernen Sie die Waschmedien. Wiederholen Sie die Wäsche noch einmal mit komplettem RPMI-Medien. Resuspendieren Pellets in bis 4 ml Medien, die meist kleinen Inseln und Kanälen (Abbildung 1D) enthält. </li><li> Hand holen die kleinen Inseln, wie in Abschnitt 5.1 beschrieben. </li></ol></li></ol> 6. GSIS Assays in isolierten Inseln <ol><li> Inkubieren der Inseln aus Schritt 5.1.4 in kompletten RPMI-Medium für 2 h in einem 37 ° C Gewebekultur-Inkubator. </li><li> Verwenden Sie eine Pipette RPMI unter einem Binokular zu entfernen. In 3 ml KRB - Lösung mit 2,8 mM Glucose zu der Platte 3. Swirl 10-mal die Inseln zu waschen. Entfernen KRB-Lösungen. <ol><li> Wiederholen Sie einmal, um den Waschvorgang. Inkubieren der Inselchen mit 3 ml KRB-Lösung bei 37 ° C für 1 h in einem Inkubator. </li></ol></li><li> Aliquot 1 ml KRB in jede Vertiefung einer 12-Well-Platte. Pre-warm die KRB Lager und Platte auf 37 ° C in einem Inkubator. </li><li> Waschen Sie die kleinen Inseln zweimal mit vorgewärmten KRB wie in 6.2. Transfer 10 Inselchen in jede Vertiefung der vorgewärmten Platten. Inkubieren in einem 37 ° C Inkubator. </li><li> Nach 45 Minuten wurden 50 ul Überstand zurücktreten. Diese enthält die Insulinlösung unter basalen Glukose ausgeschieden. </li><li> In 32 &amp; mgr; l 500 mM Glucose. Schwenken Sie die Platte 20-mal die Lösung zu mischen. Inkubieren in einem 37 ° C Inkubator. Schwenken Sie die Platte 5 mal einmal alle 5 min. </li><li> Nach 45 min zurückziehen 50 &amp; mgr; l Überstand. Dies ist das Insulin unter hohem Glucose sekretiert. </li><li> Übertragen Sie alle Inseln in ein 1,5 ml-Röhrchen. Einfrieren in -20 ° C für 30 min. Tauen Sie für 5 Minuten bei Raumtemperatur. Wiederholen Sie das Einfrieren und Auftauen Prozess. </li><li> In 0,5 ml 70% Ethanol mit 20 mM HCl. Lassen Sie über Nacht. Dies ist das Insulin links in Inselchen. </li><li> Verwenden herkömmlicher ELISA auf den Insulinspiegel Assay, zuvor beschriebenen 3. </li></ol>

<ol>
	<li>Matsumoto, S., 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=Improvement+of+pancreatic+islet+cell+isolation+for+transplantation.">Improvement of pancreatic islet cell isolation for transplantation.</a> Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. (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. M., 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=Global+gene+expression+profiling+of+pancreatic+islets+in+mice+during+streptozotocin-induced+beta-cell+damage+and+pancreatic+Glp-1+gene+therapy.">Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced beta-cell damage and pancreatic Glp-1 gene therapy.</a> Dis Model Mech. 6 (5), 1236-1245 (2013).</li><li>London, N. J., Swift, S. M., Clayton, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+functional+evaluation+of+islets+of+Langerhans.">Isolation, culture and functional evaluation of islets of Langerhans.</a> Diabetes Metab. 24 (3), 200-207 (1998).</li><li>Rorsman, P., 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=Failure+of+glucose+to+elicit+a+normal+secretory+response+in+fetal+pancreatic+beta+cells+results+from+glucose+insensitivity+of+the+ATP-regulated+K++channels.">Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.</a> Proc Natl Acad Sci U S A. 86 (12), 4505-4509 (1989).</li><li>Hellerstrom, C., Swenne, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+maturation+and+proliferation+of+fetal+pancreatic+beta-cells.">Functional maturation and proliferation of fetal pancreatic beta-cells.</a> Diabetes. 40 (Suppl 2), 89-93 (1991).</li><li>Blum, B., 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=Functional+beta-cell+maturation+is+marked+by+an+increased+glucose+threshold+and+by+expression+of+urocortin+3.">Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3.</a> Nat Biotechnol. 30 (3), 261-264 (2012).</li><li>Hegre, O. D., 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=Nonenzymic+in+vitro+isolation+of+perinatal+islets+of+Langerhans.">Nonenzymic in vitro isolation of perinatal islets of Langerhans.</a> In Vitro. 19 (8), 611-620 (1983).</li><li>Dolen&#353;ek, J., Rupnik, M. A., Sto&#382;er, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+and+differences+between+the+human+and+the+mouse+pancreas.">Structural similarities and differences between the human and the mouse pancreas.</a> Islets. 7 (1), e102445 (2015).</li><li>White, J., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Source Book of Enzymes, Source Book of Enzymes. , (1997).</li><li>Bock, T., Pakkenberg, B., Buschard, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+the+size+and+structure+of+the+endocrine+pancreas.">Genetic background determines the size and structure of the endocrine pancreas.</a> Diabetes. 54 (1), 133-137 (2005).</li><li>Bouwens, L., Rooman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+beta-cell+mass.">Regulation of pancreatic beta-cell mass.</a> Physiol Rev. 85 (4), 1255-1270 (2005).</li></ol>

The authors declare no competing financial interests for the described work.

Hier bieten wir eine Schritt-für-Schritt-Protokoll auf Inselisolierung von Bauchspeicheldrüsen, die für konventionelle Perfusion zu klein sind. Es wird erwartet , Inselchen bereit für alle islet-basierten Untersuchungen wie beta-Zellreinigung, Genexpressionsanalyse, islet beta-Zell - Reifung, Proliferation, Zellstressreaktionen, Zellüberleben, Metabolismus und funktionelle GSIS Wartung, usw. zu ergeben. Dies wird nach bestem Wissen unseren Erkenntnissen die erste detaillierte visuelle Protokoll, das neue F...

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.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<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

Perfusions-basierte Inselisolierung Protokolle von großen Säugetieren Bauchspeicheldrüsen sind gut etabliert. Solche Protokolle sind in vielen Laboratorien aufgrund der großen Größe der Pankreasgang, der ermöglicht bereit Kollagenase Injektion und anschließende Gewebeperfusion leicht durchgeführt. Im Gegensatz dazu Inselisolierung von kleinen Bauchspeicheldrüsen, wie die von neugeborenen Mäusen, ist schwierig, weil die Perfusion nicht leicht erreichbar in dem kleinen pancreata ist. Hier haben wir ein detailliertes einfaches Verfahren beschreiben, die von neu geborenen Mäusen, die mit visuellen Unterstützung einer beträchtlichen Zahl von Inseln erholt. Frisch sezierte ganze Bauchspeicheldrüsen wurden mit 0,5 mg / ml Kollagenase IV in Hanks &#39;Balanced Salt Solution (HBSS) bei 37 ° C, in Mikrozentrifugenröhrchen gelöst verdaut. Die Röhrchen wurden regelmäßig zu unterstützen Gewebe Zerstreuung angezapft. Wenn der größte Teil des Gewebes zu kleinen Gruppen etwa 1 mm dispergiert, drei- bis viermal mit Nährmedien Lysate wurden mit 10% fötalem Rinderserum (FBS), gewaschen. Islet Cluster,ohne erkennbare acinar Gewebe kann dann unter Sezieren Stereoskop zurückgewonnen werden. Dieses Verfahren gewinnt 20 - 80 kleine bis große Inseln pro Bauchspeicheldrüse von Neugeborenen Maus. Diese Inseln sind für die meisten denkbaren Downstream-Assays, einschließlich der Insulinsekretion, Genexpression und Kultur. Ein Beispiel für die Insulinsekretion Assay dargestellt, um den Isolationsprozess zu validieren. Der genetische Hintergrund und der Grad der Verdauung sind die größten Faktoren, die Ausbeute zu bestimmen. Frisch mit hoher Aktivität hergestellt Kollagenase-Lösung wird bevorzugt, da es in endokrin exokrinen Isolation hilft. Die Anwesenheit von Kationen [Calcium (Ca 2+) und Magnesium (Mg 2+)] in allen Lösungen und fötales Rinderserum in der Wasch- / Kommissionierung Medien sind notwendig für eine gute Ausbeute an kleinen Inseln mit der richtigen Integrität. Ein Binokular mit gutem Kontrast und Vergrößerung wird auch dazu beitragen.

Unter optimalen Bedingungen kann das vorgestellte Verfahren ergeben 20 - 80 Inseln aus jeder kleinen Maus Bauchspeicheldrüse. Diese Zahl hängt von dem genetischen Hintergrund, dem Alter der Mäuse, und die Größe der Inseln zurückgewonnen werden. Unter den üblicherweise verwendeten, CD1 aus gezüchtet und C57BL / 6J reinrassige Mäuse produzieren weniger Inselchen mit geringerer Größe als Hybrid zwischen CD1 und C57BL / 6 oder kommerziellen B6CBAF1 / J-Mäuse tun. Direkt Hand Komm...

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

Wir beschreiben ein Protokoll hier für intakte Inseln von neugeborenen Mäusen zu isolieren. Bauchspeicheldrüsen wurden teilweise mit Kollagenase, gefolgt von Waschen und Hand picking verdaut. 20-80 islet Cluster können pro Bauchspeicheldrüse von neugeborenen Mäusen erhalten werden, die für mehrere islet Studien geeignet sind.

Effektive Isolation von funktionellen Inselchen von Neonatal Maus Pancreas

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

effective-isolation-of-functional-islets-from-neonatal-mouse-pancreas

Research

JoVE Journal

Developmental Biology

10.0K Aufrufe.  Vanderbilt University Medical School.  Wir beschreiben ein Protokoll hier für intakte Inseln von neugeborenen Mäusen zu isolieren. Bauchspeicheldrüsen wurden teilweise mit Kollagenase, gefolgt von Waschen und Hand picking verdaut. 20-80 islet Cluster können pro Bauchspeicheldrüse von neugeborenen Mäusen erhalten werden, die für mehrere islet Studien geeignet sind. 

Serie: Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Isolierung und Kryokonservierung von neonatalen Rattenkardiomyozyten

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Forschung

Entwicklungsbiologie

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolierung und Charakterisierung von Einzelzellen aus Zebrafischembryonen

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

In - vitro - Assays für Colony Tri-potente Progenitorzellen Charakterisieren von den Adult Murine Pancreas Isoliert

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolierung von murinen embryonalen Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

Isolierung von Typ I und Typ II Perizyten aus Maus Skelettmuskeln

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Fetale kardiovaskuläre Bildgebung mit einem Hochfrequenz-Ultraschall (30 / 45MHZ) Maus

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Effiziente Generation der Bauchspeicheldrüse/Zwölffingerdarm Homeobox Protein 1+ Posterior Foregut/Bauchspeicheldrüsen-Vorfahren aus hPSCs in Haftung Kulturen

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

Isolierung spezifischer Neuronenpopulationen von Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolierung und Differenzierung von primären Myoblasten von Mausskelettmuskelexplants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Ein einfaches und effektives Transplantationsgerät für Zebrafischembryonen

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...