Bu eser CREH merkezi NIEHS tarafından ve Ulusal Doğa Bilimleri Foundation of China (No. 31572626) tarafından desteklenen bir pilot proje hibe tarafından desteklenmiştir.

Tüm hayvan deneyleri tüm ilgili kurallar, düzenlemeler ve düzenleyici kurumlar uygun olarak idam edildi. Gösterilen iletişim kuralı rehberlik ve kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Texas A & M Enstitüsü onaylanması için genomik Tıp altında gerçekleştirildi.
1. çözüm hazırlık
<ol><li>Çift Kişilik distile H2O ile 1 x 10 x Hank'in dengeli tuz solüsyonu sulandırmak ve 4 ° C'de depolayın</li>
	<li>Yalıtım çözüm HEPES (10 mM), MgCl2 (1 mM) ve glikoz (5 mM) ekleyerek hazırlayın. PH 7.4 için ayarlamak ve buz üzerinde tutun.</li>
	<li>Collagenase tip 4 collagenase çözümden hazırlayın. Not: Özellikle, collagenase tip 4 160 mg kuru ağırlığının U içerir. collagenase tip 4 1 g 5 mL Çift Kişilik distile H2O 200 mg/mL bir konsantrasyon ile seyreltilmiş. seyreltilmiş Collagenase 2 mL 30 mL buz soğuk izolasyon çözüme eklenir ve 6 mL collagenase çözeltisi 50 mL tüp her fare için transfer edilir.</li>
	<li>Yıkama çözüm yalıtım çözüm 1 mM CaCl2 ekleyerek hazırlayın.</li>
	<li>Soğuk polysucrose/sodyum diatrizoate çözüm (1.1119 g/mL, 5 mL) ile arıtma çözüm hazırlamak.</li>
	<li>Adacık kültür ortamının 500 mL L-glutamin (20 mM), penisilin (100 U/mL), streptomisin (100 µg/mL) ve fetal Sığır serum (FBS) (% 10) RPMI orta ekleyerek hazırlayın.</li>
	<li>DCFH-DA hisse senedi çözüm toz 200 µM, distile su ve store-20 ° C'de çözülerek hazırlamak Not: Çözüm boyama ROS taze DCFH-DA hisse senedi eriyik-e doğru RPMI içinde 5 mikron son konsantrasyonu sulandrarak yapılır. Bu reaktif ışıktan korunmalıdır.</li>
</ol>2. cerrahi hazırlık
<ol><li>İntraperitoneally (IP) bir fare ile avertin (%2.5 avertin, 0.4 mL/20 g) enjekte et. Sonra fare tamamen anestezi ve artık yanıt hind için pinch ayak, fare ötenazi ve biyolojik hood taşır.</li>
	<li>Karın yüzü yukarı bakacak şekilde euthanized fareyi getirin ve sterilizasyon için % 70 etanol ile cilt sprey.</li>
	<li>Karın üst karın üzerine 1 cm U-kesi ile açın ve karın boşluğu ortaya çıkarmak için göğüs üzerinde cilt rostral yönde geri çek.</li>
</ol>3. pankreas perfüzyon ve kaldırma
<ol><li>Şekil 1' de gösterildiği gibi ampulla konumunu bulmak. Duodenum Oddi Sfinkter konumunu yakın kelepçe için Kıvrımlı pensler bir çift kullanın. Bir çift daha Kıvrımlı pensler duodenum safra yolu engellemek için oniki parmak bağırsağı duvar kelepçe için kullanın. Not: Ampulla Bu adımda eklenen değil. Bu adımı duodenum safra yolu engellemek içindir.</li>
	<li>Fareyi başından proksimal ve distal açısından araştırmacı ayak yeniden konumlandırın.</li>
	<li>Ortak safra kanalını bulmak ve yavaş yavaş 30 G iğne ve 5 mL şırınga ile 3 mL collagenase çözeltisi enjekte. Ortak safra kanalını ve pankreas enjeksiyon sonra şişirilir emin olun.</li>
	<li>Şişmiş pankreas inen kolon, bağırsak, mide ve dalak ayırmak için Kıvrımlı pensler ve iyi makas kullanarak kaldırın. Pankreas 3 mL collagenase çözeltisi içeren bir 50 mL tüp içinde yerleştirin ve buz üzerinde tutun.</li>
</ol>4. pankreas sindirim
<ol><li>Her 50 mL tüp 37 ° C su banyosu yerleştirin. 15-20 dk için kuluçkaya (süre değişir zorlanma ve fareler yaş ile).</li>
	<li>Bir girdap kullanarak boru titremek.</li>
	<li>4-5 büyük steril lavabo süzgeç sindirilir pankreasta havuz ve iyice darmadağın pankreas yapmak için bir kaşık kullanabilirsiniz. Zorla ekrandan sindirilir hücreleri pipette için yıkama solüsyon içeren 23 G iğne ve 10 mL şırınga kullanın. 50 mL tüp yıkama çözümde toplamak.</li>
	<li>Tüpler, 97 x g 4 ° c için 1 dk santrifüj kapasitesi ve süpernatant dikkatle boşaltmak.</li>
	<li>Tüpler için 25 mL yıkama çözeltisi ekleyin ve doku nazik vortexing tarafından yeniden askıya alma. Spin aşağı doku, 97 x g 1 dk. için 4 ° C'de ve süpernatant dikkatle boşaltmak. İçin 3 kez tekrarlayın. Pelleted doku çıkarmak değil dikkatli olun.</li>
</ol>5. arıtma adacıkları
<ol><li>Arıtma Çözüm 10 mL yıkanmış doku Pelet ekleyin ve hafifçe içeriği resuspend.</li>
	<li>Yavaşça 5 mL yalıtım çözeltisi arıtma çözüm üzerinde yerleşimi. İki çözüm arasında keskin bir sıvı arayüzü tutmak için dikkatli olun.</li>
	<li>Tüp 4 ° C'de 15 dakika yavaş ivme ile de g x 560 ve fren yok santrifüj kapasitesi. Pipet arayüzü içine yerleştirin ve adacıkları katman yeni 50 mL tüpler içine toplamak.</li>
	<li>Adacıkları 3.5 adımda anlatıldığı gibi yıkayın.</li>
</ol>6. kaplama ve adacık hücreleri tedavi
<ol><li>Adacıkları adacıkları kültür çözüm 10 mL resuspend, yavaşça karıştırın ve 100 µL/iyi bir 96-şey plaka için çok kanallı pipet ile yerleştirin. 96 başına yaklaşık 5 adacıktan de yerleştirin.</li>
	<li>12 h için doku kültürü kuluçka plaka yerleştirin.</li>
	<li>96-şey plaka (112 x g) santrifüj kapasitesi ve kültür orta kaldırın.</li>
	<li>Çevresel kimyasallar adacıkları kültür orta sulandırmak. Her birinin kimyasal seyrelticiler 100 µL plaka ayrı kuyu ekleyin. Her iyi adacıkları resuspend ve 12 h (konsantrasyonu farklı çevresel kimyasallar için değişir) 37 ° C'de için kuluçkaya.</li>
</ol>7. ROS boyama ve ölçüm
<ol><li>1 x PBS ile adacıkları tedaviden sonra yıkayın (her 96-kuyu 200 µL). Not: Kimyasal konsantrasyonu olarak takip olduğunu: AFB1: 3 µM, Atrazine: 100 µM, BaP: 10 µM, BPA: 100 µM, β-östrojen, PCB126: 10 µM, Nonylphenol ve BPA: 100 µM.</li>
	<li>N-asetil sistein (NAC) bir antioksidan ROS gidermek için kullanın. NAC 5 mM ROS kaynaklı kimyasal seyrelticiler ekleyin ve 37 ° C'de 12 h için kuluçkaya</li>
	<li>96-şey plaka 112 x g, santrifüj kapasitesi ve 60 dk 37 ° C'de 5 µM çözüm floresan inceleyebilirsek (H2-DCFH-DA RPMI ortamda çözünmüş) ile kuluçkaya.</li>
	<li>3 kez PBS ile tedavi adacık hücreleri yıkama ve floresan 488 bir Mikroplaka Okuyucu ile ölçmek nm. Not: DCF hücrelerdeki birikimi artış tarafından 530, floresans içinde ölçülen örnek 488 heyecanlı zaman nm nm.</li>
	<li>Glikoz uyarılmış insülin salgılanması (GSIS) tahlil9 xenobiotic ROS-inducing gerçekleştirmek tedavi adacıkları ve tedavi edilmezse adacıkları pankreas adacıkları fizyolojik fonksiyonu xenobiotic etkisini ölçmek için.</li>
</ol>

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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Kanıt biriken öneriyor çevresel kimyasallar o maruz T2DM gelişiminde önemli bir rol oynar. Xenobiotics kaynaklı ROS T2DM gelişmesine katkıda bulunan bir potansiyel etyolojik faktör olarak kabul edilmiştir. İnsanlar xenobiotic kimyasalların geniş bir dizi sunulur ve bir büyük gerek roman araştırma teknikleri etkili pankreas toxicants tanımlamak ve toksisite pankreas hücreleri belirli mekanizmasının araştırmak için.
Bu çalışmada, yayımlanmış yordamlar14, dayalı geliştirdiğimiz fare ve ekran pankreas adacıkları yalıtmak için bir protokol oksidatif stres kaynaklı zarar pankreas hücreleri için xenobiotic kimyasallar içeren hücreleri. Ayrıca xenobiotic bileşikler tarafından indüklenen pankreas belirli toksik yanıtlar araştırmak için bu yordamı kullanın. Pankreas fareden elde etmek için kan şekeri düzeylerini değiştirmez ve pankreas15,16,17damarlara etkilemez çünkü diğer anestezi üzerinde avertin kullanmayı tercih. Bulduk yoğunluk gradient Santrifüjü adım adacık yüksek saflıkta ile yalıtım için önemlidir. Enkaz ve ekzokrin hücreler/doku yoksun bir saf adacıkları örnek almak için polysucrose/sodyum ve yalıtım arabellek faz arasında farklı katman elde etmek için özen gösterilmelidir. Yöntem pankreas adacık fizyolojisi, GSIS tahlil gibi çevresel kimyasal etkileri vitro analiz için ( Şekil 3' te gösterildiği gibi) kullanılabilir. Adacıkları dikkatli 96-hücre kültürü plaka üniformalı cep numarası sağlamak için ayrı hücrelere tekrarlanan pipetting ile dağınık. Yöntem tanımlamak burada yalıtım adacık ve hızlı bir tahlil ROS üretimi için kombine ve bu nedenle, potansiyel pankreas zarar veren kimyasallar için basit ve etkili başlangıç ekranı.
Biz insülin salgılanması yanıt glikoz meydan okuma olarak izole pankreas adacıkları kimliğini onaylamak için kullanılır. Yüksek insülin salgılanması (Şekil 3) ve kamp üretimi, xenobiotic tedaviler18yanıt dahil olmak üzere Parametrelerindeki değişiklikler çözümlenmesi için uyarlanabilir bir yöntemdir.

T2DM yaygınlığı artışlar küresel sağlık krizi son yıllarda poz halk sağlığı1için ciddi bir tehdit haline gelmiştir. Pek çok faktör bulunmuştur nedensel T2DM, geliştirme arasında hangi, bağlanacak şekilde yinelenen bulgular bu faktörler için bir ortak yakınsak noktası aşırı ROS2 nesil uzaklaşan oksidatif stres indüksiyon olduğunu göstermektedir , 3.
Çevresel kimyasallar PCB, dioksin ve BaP gibi geniş bir yelpazede pankreas β hücrelerinin işlevini bozabilir ve insülin direnci ve T2DM4yol oksidatif stres, neden tespit edilmiştir. Her ne kadar ROS fizyolojik düzeyi hücresel işlevlerinde önemli bir rol oynar, antioksidan sistem kapasitesini aşıyor ROS maruz hücreler/dokulara zarar sonuçları ve hastalıklar5' e götürür. Pankreas Beta hücreleri antioksidan enzim düşük düzeyde hızlı ve dolayısıyla oksidatif stres-aracılı hasar6,7için önemli bir hedef. Kronik ROS yüksek düzeyde maruz stres kaynaklı pankreas hücresi disfonksiyon5 neden için gösterilen yanı sıra karaciğer ve yağ dokusu8insülin direnci.
Bu projenin genel amacı bir hücre tabanlı tahlil ekran kimyasallara ROS pankreas hücreleri içinde onların indüksiyon dayalı onların diyabetojenik potansiyeller için geliştirmektir. Pankreas ve metabolik detoksifikasyon yoksun xenobiotic indüklenen zarar6,7için önemli bir hedeftir. Bu nedenle, doğrudan pankreas hücrelerinde üretilen ROS ölçerek, bu tahlil pankreas kimyasal kaynaklı yaralanma doğrudan bir yaklaşım sağlamalıdır. Bu yöntem geliştirmek için biz fare pankreas adacıkları izole, izole adacık hücre kültür koşul kimyasallar ile altında kültürlü ve kimyasal kaynaklı ROS üretimi okuma olarak kullanılmaktadır. Bu basit ve izole adacık kimyasal maddeler ROS-inducing belirlenmesinde etkili işlemdir; soruşturma için pankreas içinde vitroözgü mekanizmaları toksisite için daha fazla geliştirilebilir.

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

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

Bazı çevresel kimyasallar insan ve hayvanlarda maruz hangi-ecek götürmek tip 2 diabetes mellitus (T2DM) gelişimi için pankreas β hücrelerinin hücre hasarına neden tespit edilmiştir. Kimyasal kaynaklı β hücre hasarı için mekanizmalar belirsiz ve karmaşık olması olasılığı olmasına rağmen bir yinelenen bu kimyasalların oksidatif stres zarar neden aşırı reaktif oksijen türleri (ROS) üretimi için önde gelen neden bulgudur β hücre. Potansiyel diyabetojenik çevresel kimyasallar tanımlamak için C57BL/6 fareler pankreas adacık hücreleri ve kültürlü adacık hücreleri 96-şey hücre kültür tabak içinde izole; daha sonra adacık hücreleri kimyasallar ile ilaç ve ROS üretimi 2' tarafından 7'-dichlorofluorescein (DCFH-DA) floresan boya tespit edildi. Bu yöntemi kullanarak, biz o Bisfenol A (BPA), Benzo [a] dumanında (BaP), Poliklorlu bifeniller (PCB) bulup, potansiyel olarak adacık hücreleri hasara neden olabilir düşündüren ROS, yüksek düzeyde neden. Bu yöntem diyabetojenik xenobiotics eleme için yararlı olmalıdır. Buna ek olarak, kültürlü adacık hücreleri de kimyasal kaynaklı toksisite pankreas hücreleri vitro analiz için adapte olabilir.

Sağlıklı izole adacık test adacıkları olan bir yuvarlak veya oval şekil (boyutu tekdüzelik zorlanma zorlanma değişebilir rağmen) nispeten boyutu üniforma olduğu Şekil 2' de gösterilmiştir. Biz sonraki adacık yalıtma ve kültür adacıkları insülin salgılanmasını uyarıcı bir vitro tahlil pankreas adacık işlevlerinde araştırdık. Şekil 3 C57BL/6 izole fare adacıkları 3,3 mM ve 16,7 mM glikoz9tarafından indüklenen GSIS tahlil tipik bizim analizini gösterir.
Plaka format ekran yöntemi kullanarak, biz ROS önemli indüksiyon adacık hücreleri (Şekil 4) neden birkaç xenobiotics belirledik. Biz izole adacıkları 12 h için farklı kimyasallar ile tedavi ve o Atrazine, BPA, BaP ve PCB126bulunur ve β-östrojen ROS önemli indüksiyon izole adacıkları10,11,12 ' neden olabilir , 13.
ROS diyabetojenik indüksiyon eşit olmasa da, ROS önemli rol T2DM8' e açan fizyolojik fonksiyonları pankreas β hücrelerinin zarar olarak gösteren yeterli delil var. Bu nedenle, çalışma değeri başlangıç ekranı olarak kimyasal inducing bu ROS belirlemektir. Ayrıca, tanımlanan bileşikler ayrıca el yazması açıklanan izole pankreas adacık hücre kültür sistemi içinde analiz vitro olabilir. İnsülin salgılanmasını glikoz uyarıcı kısaca diyabetojenik veri sağlamak için test edilmiş bu bileşiklerin izole adacıkları üzerinde ROS-inducing xenobiotics etkilerini göstermiştir. Pankreas adacıkları PCB126 ve BPA ile önceden tedavi insülin salgısını kontrol grubu (Şekil 5) daha önemli bir azalma gösterdi.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57327/57327fig1v3.jpg"> Resim 1 : Fare pankreas perfüzyon prosedürü. Yordamı aşağıdaki adımlardan oluşur: (1) ampulla bulmak ve ampulla kelepçe için iki çift eğri forseps kullanın. (2) ortak safra aracılığıyla iğne yerleştirin. (3) yavaş yavaş 3 mL 5 mL şırınga collagenase çözüm stokta enjekte et. (4) oniki parmak bağırsağı--dan başlayan şişmiş pankreas toplamak. 
 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57327/57327fig2v3.jpg"> Resim 2 : İzole fare adacıkları. Kültür ortamında izole fare pankreas adacıkları gösterilir. 500 µm ölçek çubuğu.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57327/57327fig3v3.jpg"> Şekil 3 : Glikoz uyarılmış insülin salgılanması izole pankreas adacıkları. Doz bağımlı glikoz uyarılmış insülin salgılanması (1, 3.3 ve 16,7 mM, tedavi için 15 dk) bir fare insülin ELISA kullanarak (ortalama 5 adacıktan iyi başına) izole adacıklar içinde ölçüldü kiti. Hata çubukları standart sapmalar gösterir. p < 0,05.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57327/57327fig4v3.jpg"> Şekil 4 : ROS xenobiotics C57BL/6 fareler gelen izole adacıklar içinde tarafından indüksiyon. (A)izole adacıkları 12 h için tedavi ve floresan boya 2', 7'-dichlorofluorescein (DCFH-DA) 1 h için lekeli. Floresan bir floresan plaka okuyucu tarafından tespit edilmiştir. (B) kimyasal kaynaklı ROS NAC (5 mM) tarafından su. (AFB1: 3 µM, Atrazine: 100 µM, BaP: 10 µM, BPA: 100 µM, PCB126: 10 µM, Nonylphenol, BPA: 100 µM ve NAC: 5 mM). Hata çubukları standart sapmalar gösterir. p < 0,05. <a href="/files/ftp_upload/57327/57327fig4v3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57327/57327fig5v3.jpg"> Şekil 5 : Xenobiotic glikoz uyarılmış insülin salgılanması izole adacıklar etkisi. İzole adacıkları PCB126, β-östrojen ve BPA (4 h) ile önceden tedavi ve adacık kültür orta 1 h için tuttu. Süpernatant adacıklar insülin bir ticari olarak mevcut fare insülin ELISA tarafından ölçüm için toplanan tahlil. Hata çubukları standart sapmalar gösterir. p < 0,05.

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

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

Burada potansiyel diyabetojenik xenobiotic kimyasallar tanımlamak için xenobiotics tarafından ROS indüksiyon süzmek için fare pankreas adacık hücreleri izole etmek için bir iletişim kuralı mevcut.

Bir fare pankreas adacık hücre tabanlı tarama diyabetojenik çevresel kimyasallar için

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

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K Görüntüleme.  Hunan Agriculture University. Burada potansiyel diyabetojenik xenobiotic kimyasallar tanımlamak için xenobiotics tarafından ROS indüksiyon süzmek için fare pankreas adacık hücreleri izole etmek için bir iletişim kuralı mevcut.

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

Murine Pancreatic Islet Isolation

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

Murin Pankreas Adacık İzolasyonu

Biyoloji

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

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

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

Fare pankreatik endokrin hücreleri tek hücre Transcriptomic analizleri

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

Gelişim Biyolojisi

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

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

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

İnsan Dışı Primat Pankreatik Adacık Oksijen Tüketiminin Analizi

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

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

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

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

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

Lepr<sup&gt; Db</sup&gt; Tip 2 Diyabet Fare Modeli: Bozulmamış Islets Of Pankreas Adacık İzolasyonu ve Canlı hücre 2-Foton Görüntüleme

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

Tıp

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

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

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

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

Bir Yüksek içerik<em&gt; İn Vitro</em&gt; Pankreas Adacık β-hücre çoğaltma Keşif Platformu

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

An In Ovo Model for Testing Insulin-mimetic Compounds

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

An In Ovo Model for Testing Insulin-mimetic Compounds

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

İnsülin mimetic bileşikler test etmek için bir Ovo içinde modeli

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

Nörobilim

Intraportal Transplantation of Pancreatic Islets in Mouse Model

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

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

İntraportal fare modeli pankreas adacıkları nakli

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

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

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

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

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 ...

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 ...

Murin Pankreatik β-Hücrelerinde Mitofajinin Kantitatif Analizi

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

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

Yazar Spotlight: Pankreas β hücrelerinde mitofaji akısının etkili ve sağlam tamamlayıcı yaklaşımlar kullanılarak değerlendirilmesi 

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

Pankreas β Hücrelerinde Mitofaji Akısını Sorgulamak İçin Tamamlayıcı Yaklaşımlar

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

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