Unterstützt von der Gründung an der University of Illinois at Chicago sowie Inselzell Resource Center NIH (RFA-RR 05 bis 003), der Christopher-Stiftung, die Efroymson Foundation, und die Tellabs-Stiftung.

1. Reinigung von kleinen Inseln <ol><li> So starten Sie die Insel Reinigungsverfahren, laden Sie die COBE Tasche, klemmen alle Rohre mit Ausnahme der grünen Röhre, und die Geschwindigkeit auf 1.500 und der Super-out auf 0 gesetzt. </li><li> In der Haube Einrichten der Pumpe und Gradienten Bechern und verbinden Sie die Sammlung Schlauch an die grüne Röhre. </li><li> Nun ist die Belastung des Ficoll 1,100 kann beginnen: push &quot;spin&quot; fügen Sie dann 110 ml Ficoll 1,100 bis vorne Becher, und starten Sie die Pumpe, um die COBE Tasche zu laden. Da die Ficoll eingelegt ist, drücken Super Out, stoppen Sie die Pumpe, ausspannen Pumpenkopf und stellen Sie die Super-out Geschwindigkeit auf 100. </li><li> Wenn die Ficoll das Becherglas erreicht und alle Luft aus dem Schlauch, re-Klemme am Pumpenkopf vertrieben. Stellen Sie die Schleuderdrehzahl bis 3.000, und der Super-out zu 0 aus. </li><li> Nun bereiten die Gradienten durch Eingießen der schweren Steigung in den vorderen Becher zu laden. Leicht Release der Klemme zwischen den beiden Bechern, um die Luft zu entfernen. Dann gießen Sie den leichten Steigung in den Rücken Becher. </li><li> Schalten Sie den Magnetrührer, drücken Sie auf &quot;Spin&quot; auf dem COBE-Maschine, entnehmen Sie die Klemme den Schlauch zwischen den beiden Bechern und visuell überprüfen, ob die schweren und leichten Steigungen mischen. </li><li> Sobald die Gradienten begonnen Mischen, auf der Zentrifuge drehen. Warten Sie eine Minute, und dann an der Pumpe drehen, um die Vermischung Gradienten zu laden. </li><li> So laden Sie das Gewebe lassen die Steigung gering laufen in den vorderen Becher, aber nicht niedrig genug, damit die Luft, um das Laden Rohr geben, dann umzuspannen das Verbindungsrohr die Becher und laden das Gewebe. </li><li> Weiter Hinzufügen der Rest des Gewebes. Dann waschen die Röhre, die das Gewebe mit 50 ml Waschlösung gehalten, und es verwenden, um den vorderen Becher zu waschen. </li><li> Als letzter des Gewebes in die Tasche, stop gleichzeitig die Pumpe, Ausspannen am Pumpenkopf, und klemmen Schlauch über der Tasche. Weiterhin für 5 Minuten zentrifugiert. </li><li> Während der Zentrifugation weiter, bringen den 12-Röhrchen auf der Motorhaube und lockern ihre Mützen. Bringen Sie die Sammlung Schlauch an den gelben Schlauch, und legen Sie die sterile Sammlung Spitze in konischen Rohr 1. Dann Ausspannen den gelben Schlauch, und klemmen Sie die grüne Röhre. </li><li> Am Ende der Spin, langsam erhöhen Superout bis 100 auf. Sammeln Sie 150 ml in Röhrchen 1. Dann sammeln 30 ml in Röhrchen 2-12 (von 200 bis 230 ml). </li><li> Nach der vollständigen Erfassung abgeschlossen ist, drücken Sie &quot;STOP&quot; auf dem COBE. Jetzt, Reinigung beendet ist, können die kleinen Inseln beurteilt und kultiviert werden. </li></ol> 2. Sampling und Kultur <ol><li> Nach dem Sammeln der gereinigten Inseln Fraktionen, entfernen Sie eine Probe aus jedem der 12 Sammelröhrchen und Fleck mit Dithizon. Die Färbung wird zeigen, welche Schichten enthalten hochreine Inseln vs Schichten mit geringer Reinheit Inselchen. </li><li> Sammeln Sie die hohen und niedrigen Reinheit Gewebe und Pool jeweils zusammen. </li><li> Spin und waschen zweimal. Nach dem zweiten Waschen, bringen die Lautstärke der einzelnen zu 250 ml mit Final Wash Medien. </li><li> Zur Beurteilung der gepoolten Fraktionen beginnen, indem sie zwei 1 ml-Proben aus dem hohen der vereinigten Fraktionen und einer 1 ml Probe aus dem niedrig. Übertragen Sie jede 1ml Probe in einem konischen Rohr mit 9 ml Waschlösung. </li><li> Dann je 1 ml von je 15 ml Tube zu einem Zählschale. Untersuchen und zählen Sie die Zellen unter dem Mikroskop. </li><li> Nach Auszählung der Zellen zu berechnen die Anzahl der Flaschen benötigt, um Kultur der Inseln mit folgender Formel: (insgesamt EIN / Reinheit der Inseln in dezimal) / (30.000 EIN pro Flasche). Zum Beispiel sind 100.000 Inseln mit 90% Reinheit in 4-Kolben kultiviert. </li><li> Übertragen Sie die Inseln zu T175 Flaschen und Kultur in einem 37 ˚ C und 5% CO 2-Inkubator. </li></ol>

<ol>
	<li>Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Nano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+for+allotransplantation:+variables+associated+with+successful+islet+yield+and+graft+function.">Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.</a> Diabetologia. 48, 906-912 (2005).</li><li>Barbaro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84, 1200-1203 (2007).</li><li>Lakey, J. R., Rajotte, R. V., Warnock, G. L., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pancreas+preservation+prior+to+islet+isolation.+Cold+ischemic+tolerance.">Human pancreas preservation prior to islet isolation. Cold ischemic tolerance.</a> Transplantation. 59, 689-694 (1995).</li><li>Fridell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+histidine-tryptophan-ketoglutarate+solution+and+University+of+Wisconsin+solution+for+organ+preservation+in+clinical+pancreas+transplantation.">Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation.</a> Transplantation. 77, 1304-1306 (2004).</li><li>Potdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+experience+using+histidine-tryptophan-ketoglutarate+solution+in+clinical+pancreas+transplantation.">Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation.</a> Clin Transplant. 18, 661-665 (2004).</li><li>Salehi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+isolation+outcomes+from+pancreata+preserved+with+Histidine-Tryptophan+Ketoglutarate+versus+University+of+Wisconsin+solution.">Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution.</a> Transplantation. 82, 983-985 (2006).</li><li>Ricordi, C., Lacy, P. E., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+islet+isolation+from+human+pancreas.">Automated islet isolation from human pancreas.</a> Diabetes. 38 Suppl 1, 140-142 (1989).</li><li>Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+assessment+in+man+and+large+animals.">Islet isolation assessment in man and large animals.</a> Acta Diabetol Lat. 27, 185-195 (1990).</li></ol>

Trotz erheblicher Fortschritte in der Technik der menschlichen Inselzellen isoliert bleiben Insel Ertrag sehr variabel und unvorhersehbar. Die Reinigung der verdauten Pankreasgewebe ist entscheidend, um eine ausreichende Insel Masse nach erfolgreicher enzymatische Verdauung für die Transplantation zu erholen. Die UIC Reinigungsverfahren wird empfohlen, da eine höhere Gewinnung von hochreiner menschlichen Inselchen mit dieser Methode nachgewiesen wurde. Darüber hinaus können bis zu 50 ml verdaute Gewebe in einem einz...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman Coulter</td>
<td>424803</td>
<td>&nbsp;</td>
<tr>
<td>Cobe 2991 Cell Processor</td>
<td>equipment</td>
<td>Gambro. BCT</td>
<td>2556</td>
<td>islet purification</td>
</tr>
<tr>
<td>MZ6 Inverted Microscopy TLBD4.1</td>
<td>equipment</td>
<td>Leica Diagnostic Instrument Co</td>
<td>4103</td>
<td>&nbsp;</td>
<tr>
<td>Cell Culture Dish with 2 mm Grid</td>
<td>equipment</td>
<td>Nunc Brand Products</td>
<td>174926</td>
<td>islet counting</td>
</tr>
<tr>
<td>Digital Sight DS L1</td>
<td>equipment</td>
<td>Nikon</td>
<td>217267</td>
<td>&nbsp;</td>
<tr>
<td>COBE bag</td>
<td>disposable equipment</td>
<td>O.R. Solution</td>
<td>66707003</td>
<td>&nbsp;</td>
<tr>
<td>T175 culture flask</td>
<td>disposable equipment</td>
<td>Sarstedt</td>
<td>83.1812.500</td>
<td>&nbsp;</td>
<tr>
<td>University of Wisconcin (UW) solution</td>
<td>reagent</td>
<td>DURAMED</td>
<td>1000-46-06</td>
<td>&nbsp;</td>
<tr>
<td>Hank's Buffered Salt Solution (HBSS)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-597-CM</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll 1.100</td>
<td>reagent</td>
<td>Bichrom AG</td>
<td>L6155</td>
<td>&nbsp;</td>
<tr>
<td>M199 media (wash solution)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-784-CM</td>
<td>&nbsp;</td>
<tr>
<td>Final wash/Culture Medium</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-785-CV</td>
<td>&nbsp;</td>
<tr>
<td>Dithizone</td>
<td>reagent</td>
<td>Sigma </td>
<td>D5130</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

human pancreatic islet isolation: part ii: purification and culture of human islets

Management von Typ 1 Diabetes ist belastend, sowohl für den Einzelnen und die Gesellschaft, im Wert von über 100 Milliarden Dollar jährlich. Trotz der weit verbreiteten Verwendung von Glukose-Monitoring und neue Insulin-Formulierungen, viele Menschen immer noch zu entwickeln verheerenden Folgekomplikationen. Inselzellen der Bauchspeicheldrüse Transplantation kann bei normalen Blutzuckerspiegels bei Diabetes-Patienten wiederherstellen<sup&gt; 1</sup&gt;, Ohne das Risiko schwerer Hypoglykämien, die mit einer intensivierten Insulintherapie assoziiert sind. Die ausreichende Insel Masse ist für eine erfolgreiche Inseltransplantation wichtig. Allerdings wirken sich Spender Eigenschaften, Orgel Beschaffung und Erhaltung der Isolation Ergebnis<sup&gt; 2</sup&gt;. An der University of Illinois at Chicago (UIC) entwickelten wir eine erfolgreiche Isolierung Protokoll mit einer verbesserten Reinigung Gradienten<sup&gt; 3</sup&gt;. Das Programm startete im Januar 2004 und mehr als 300 Isolierungen wurden bis November 2008 durchgeführt. Der Bauchspeicheldrüsen wurden in kaltem Konservierungslösungen geschickt (UW, University of Wisconsin-oder HTK, Histidin-Tryptophan Ketoglutarat)<sup&gt; 4-7</sup&gt; Zu den Cell Isolation Laboratory an UIC für Inselzellen isoliert. Langerhans-Inseln wurden isoliert mit der UIC-Methode, die eine modifizierte Version der Methode ursprünglich von Ricordi beschrieben wird<em&gt; Et al</em&gt;<sup&gt; 8</sup&gt;. Wie in Teil I: Die Verdauung und Sammlung von Pankreasgewebe wurde menschlichen Pankreas getrimmt, durchbohrt, perfundierten und verdaut. Nach dem Sammeln und mindestens 30 Minuten Inkubation in UW-Lösung wurde das Gewebe in der Feldtrenner geladen (COBE 2991, Cobe, Lakewood, CO) für die Reinigung<sup&gt; 3</sup&gt;. Nach der Reinigung, Inselchen Ausbeute (ausgedrückt als Insel-Äquivalenten, IEQ), Gewebe Volumen und Reinheit wurde nach Standard-Methoden ermittelt<sup&gt; 9</sup&gt;. Isolierte Inseln wurden in CMRL-1066 Medien (Mediatech, Herndon, VA), mit 1,5% Humanalbumin, 0,1% Insulin-Transferrin-Selen (ITS), 1 ml Ciprofloxacin, 5 ml 1M HEPES ergänzt, und 14,5 ml von kultivierten 7,5% Natriumbicarbonat in T175-Kolben bei 37 ° C über Nacht Kultur vor Inselchen transplantiert wurden oder für Forschungszwecke verwendet.

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets at JoVE.com

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

Das Erreichen hoher Qualität und ausreichender Menge menschlicher Inselchen ist einer der führenden Voraussetzungen für eine erfolgreiche Inseltransplantation. In diesem Video, beschreiben wir Schritt für Schritt die Verfahren für die menschliche Inselzellen der Bauchspeicheldrüse Isolation (Teil II: Reinigung und Kultur der menschlichen Inseln) mit einer modifizierten automatisierte Methode.

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part II: Reinigung und Kultur der Menschenrechte Islets

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

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15.4K Aufrufe.  University of Illinois, Chicago. Das Erreichen hoher Qualität und ausreichender Menge menschlicher Inselchen ist einer der führenden Voraussetzungen für eine erfolgreiche Inseltransplantation. In diesem Video, beschreiben wir Schritt für Schritt die Verfahren für die menschliche Inselzellen der Bauchspeicheldrüse Isolation (Teil II: Reinigung und Kultur der menschlichen Inseln) mit einer modifizierten automatisierte Methode.

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

Murine Pancreatic Islet Isolation

Murine Inselzellen der Bauchspeicheldrüse Isolation

Forschung

Biologie

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

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

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

Menschliche Inselzellen der Bauchspeicheldrüse Isolation: Part I: Verdauung und Sammlung von Pankreasgewebe

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolierung von Stammzellen aus menschlichen Bauchspeicheldrüsenkrebs Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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.

Eine Methode zur Maus-Inselzellen der Bauchspeicheldrüse Isolation und intrazellulären cAMP-Bestimmung

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

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

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

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

Isolierung und Kultivierung von primären Maus Azinuszellen des Pankreas

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

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Effizienten Herstellung und Reinigung von rekombinantem murinem Kindlin-3 von Insektenzellen für biophysikalische Studien

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex-vivo-Kultur von embryonalen Maus-Haut-und-Live-Darstellung von Migration Melanoblasten

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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.

Ein einfaches Hocheffizienzprotokoll für die Isolierung von Pankreas-Isleten von Mäusen

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

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolierung und Anreicherung menschlicher Lungenepithel-Vorläuferzellen für Organoidkulturen

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...