Adacık Hücre Kaynak Merkezi NIH hibe (RFA-RR 05-003), Christopher Vakfı Efroymson Vakfı ve Tellabs Vakfı olarak, Chicago Illinois Üniversitesi kurucu olarak tarafından desteklenen.

1. Adacık saflaştırılması <ol><li> Adacık arıtma işlemi başlatmak için, COBE çanta yüklemek yeşil tüp hariç tüm tüpler kelepçe ve 0 1.500 için hızını ayarlamak ve süper. </li><li> Kaputun pompa ve degrade bardak kurma ve toplama boru yeşil tüpüne bağlamak. </li><li> : &quot;Spin&quot; sonra ön behere 110 ml Ficoll 1.100 itin ve COBE çanta yüklemek için pompa start Şimdi Ficoll 1.100 yükleme başlayabilirsiniz. Ficoll yüklü olduğundan, süper dışarı basın durdurmak pompa, pompa kafası çözülme ve 100 süper çıkış hızını ayarlamak. </li><li> Ficoll beher ulaşır ve boru, pompa başında yeniden-klemp tüm hava dışarı atılır. 3.000 spin hız ve süper çıkış hızı 0 olarak ayarlayın. </li><li> Şimdi ön behere ağır degrade dökerek gradyanlar yüklemek için hazırlar. Hafif herhangi bir havayı çıkarmak için iki bardak arasındaki kelepçe bırakın. Sonra ışık degrade arka behere dökün. </li><li> Manyetik karıştırıcı açın, COBE makine &quot;spin&quot; tuşuna basın, iki bardak bağlayan tüp kelepçe kaldırmak ve görsel olarak, ağır ve hafif gradyanlar karıştırma olduğundan emin olun. </li><li> Gradyanlar karıştırma başladıktan sonra, santrifüj açmak. Bir dakika bekleyin ve sonra karıştırma gradyanlar yüklemek için pompa açın. </li><li> Doku degrade ön beher düşük çalıştırmak yük, ancak hava yükleme tüp girmek için yetecek kadar düşük, daha sonra bardak bağlayan tüp reclamp ve doku yüklemek için. </li><li> Dokusunun geri kalanını eklemeye devam edin. Daha sonra 50 ml yıkama solüsyonu ile doku düzenlenen tüp yıkama ve ön beher yıkamak için kullanın. </li><li> Doku son çanta girerken, aynı anda, pompa durdurmak pompa başındaki çözülme ve yastığının üzerine boru kelepçe. 5 dakika santrifüj devam edin. </li><li> Santrifüj devam ederken, kaput için 12 toplama tüpleri getirmek ve onların kapakları gevşetin. Sarı tüp toplama boru takın ve steril toplama ucu konik tüp 1 içine yerleştirin. Sonra sarı tüp çözülme ve yeşil tüp kelepçe. </li><li> Spin sona erdiğinde, yavaş yavaş 100 Superout yükseltmek. 1 tüp 150 ml toplayın. Sonra (200-230 ml) 30 ml tüpler 2-12 toplamak. </li><li> Koleksiyonu tamamlamak, COBE &quot;DUR&quot; itin. Şimdi bu arıtma sona erdi, adacıklar değerlendirilmeli ve kültür olabilir. </li></ol> 2. Örnekleme ve kültür <ol><li> Saflaştırılmış adacıklar kesirler topladıktan sonra, her biri 12 toplama tüpleri bir örnek çıkarın ve Dithizone ile leke. Boyama katmanları düşük saflık adacıklar yüksek saflıkta adacıklar vs katmanları içeren gösterecektir. </li><li> Yüksek ve düşük saflıkta doku ve havuz her bir araya toplayın. </li><li> Spin ve iki kez yıkayın. Final Yıkama medya ile ikinci yıkamadan sonra, her birinin hacmi 250 ml getirmek. </li><li> Değerlendirmek için toplanmış fraksiyonları düşük havuzlanmış kesirler ve bir 1 ml örnek yüksek iki 1 ml örnek alarak başlar. Her 1 ml örnek 9 ml yıkama solüsyonu içeren konik bir tüp içine aktarın. </li><li> Daha sonra, her 15 ml tüp sayma çanak 1 ml transfer. Hücrelerin mikroskop altında incelemek ve saymak. </li><li> (Ondalık adacıklar toplam EIN / saflık) / (şişesi ortalama 30.000 EIN): hücreler, kültür, aşağıdaki formül kullanılarak adacıklar için gerekli şişe sayısını hesaplamak sayımından sonra. Örneğin, 100.000 adacıklar% 90 saflıkta 4 şişeler kültür. </li><li> 37 T175 matara ve kültür adacıkları transfer ˚ C ve% 5 CO 2 inkübatör. </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>

Insan adacık izolasyon teknikleri konusunda önemli gelişmeler olmasına rağmen, adacık verimi son derece değişken ve öngörülemez kalır. Arıtma sindirilir pankreas dokusu nakli başarılı enzimatik sindirim sonra yeterli bir isle kütlesi kurtarmak için çok önemlidir. Bu yöntemi kullanarak, son derece saf bir insan adacıklar üstün bir kurtarma gösterildi, çünkü UIC arıtma yöntemi tavsiye edilir. Ayrıca, 50 ml kadar sindirilmiş doku böylece insan adacıklar iskemi ile ilişkili yaralanma izola...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Tip 1 diyabet Yönetimi, yıl içinde 100 milyar dolar maliyet, hem birey ve toplum, külfetli. Glikoz izleme ve yeni insülin formülasyonları yaygın kullanımına rağmen birçok kişi hala yıkıcı ikincil komplikasyonlar gelişir. Pankreas adacık nakli, diyabetik hastalarda normal glukoz kontrolü yakın geri yükleyebilirsiniz<sup&gt; 1</sup&gt;, Yoğun insülin tedavisi ile ilişkili ciddi hipoglisemik atakların riski olmadan. Başarılı adacık nakli için yeterli adacık kitle sağlanması önemlidir. Ancak, donör özellikleri, organ temini ve korunması izolasyon sonucunu etkileyebilecek<sup&gt; 2</sup&gt;. University of Illinois at Chicago (UIC), gelişmiş bir arıtma degrade ile başarılı bir izolasyon protokolü geliştirdi<sup&gt; 3</sup&gt;. Bu program, 2004 yılı Ocak ayında başlayan ve 300&#39;den fazla izolasyonların Kasım 2008&#39;de yapıldı. Pancreata (UW, Wisconsin veya HTK Üniversitesi, Histidin-Triptofan Ketoglutarate), soğuk muhafaza çözümleri gönderildi<sup&gt; 4-7</sup&gt; Hücre İzolasyonu Laboratuvar adacık izolasyonu için UIC. Pankreas adacık Ricordi tarafından tanımlanan yöntem değiştirilmiş bir versiyonu olan UIC yöntemi ile izole edildi<em&gt; Ve ark.</em&gt;<sup&gt; 8</sup&gt;. Olarak Bölüm: Sindirim ve Pankreas Doku Koleksiyon, insan pankreas, kesilmiş delikli, perfüze ve sindirilmiş oldu. Toplama ve UW çözüm inkübasyon en az 30 dakika sonra, arıtma için doku hücre ayırıcı (2991 COBE, COBE, Lakewood, CO) yüklenmiş<sup&gt; 3</sup&gt;. Ardından arıtma, adacık verim (adacık benzerleri, iEQ olarak ifade), doku hacmi ve saflık standart yöntemlere göre belirlenir.<sup&gt; 9</sup&gt;. İzole adacıklar CMRL-1066,% 1.5 insan albümini,% 0.1 &#39;lik insülin transferrine selenyum (ITS) Siprofloksasin, 5 ml 1M Hepes, 1 ml ile desteklenen medya (Mediatech, Herndon, VA), ve 14.5 ml kültüre edildi T175 şişeler 7.5% Sodyum Bikarbonat, 37 ° C gecede kültür adacıkları nakledilen veya araştırma için kullanılan önce.

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

, Yüksek kalite ve insan adacıklar uygun miktarda sağlamak başarılı bir adacık nakli için önemli önkoşullar biridir. Bu video, adım adım tarif insan pankreas adacık izolasyonu (Bölüm II: insan adacıklar arıtma ve kültür) için prosedürleri değiştirilmiş bir otomatik yöntemi kullanarak.

İnsan Pankreas Adacık İzolasyonu: Bölüm II: İnsan Islets saflaştırılması ve Kültür

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 Görüntüleme.  University of Illinois, Chicago. , Yüksek kalite ve insan adacıklar uygun miktarda sağlamak başarılı bir adacık nakli için önemli önkoşullar biridir. Bu video, adım adım tarif insan pankreas adacık izolasyonu (Bölüm II: insan adacıklar arıtma ve kültür) için prosedürleri değiştirilmiş bir otomatik yöntemi kullanarak.

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

Murine Pancreatic Islet Isolation

Murin Pankreas Adacık İzolasyonu

Biyoloji

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.

İnsan Pankreas Adacık İzolasyonu: Bölüm I: Sindirim ve Pankreas Doku Koleksiyon

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.

İnsan Pankreas Kanseri Xenografts Kök Hücre İzolasyonu

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.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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.

Fare Pankreas Adacık İzolasyonu ve hücre içi cAMP Belirlenmesi için Bir Yöntem

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.

Fare Primer Pankreas Asiner Hücreleri İzolasyon ve Kültür

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.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

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.

Fare Embriyonik Cilt ve Melanoblast Göç Canlı görüntüleme ex vivo Kültür

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.

Farelerden Pankreas Adacık İzolasyon için Basit Bir Yüksek Verimlilik Protokolü

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.

Organoid Kültür için İnsan Akciğer Epitel Progenitör Hücrelerinin İzolasyonu ve Zenginleştirilmesi

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