وأيد هذا العمل بمنحه مشروع تجريبي من مركز كريه برعاية نييهس ومؤسسة العلوم الطبيعية الصينية الوطنية (رقم 31572626).

وقد أعدم جميع التجارب على الحيوانات امتثالا لجميع المبادئ التوجيهية ذات الصلة واللوائح والوكالات التنظيمية. وأجرى في بروتوكول يتم شرحها تحت التوجيه والموافقة على "رعاية الحيوان المؤسسية" واستخدام اللجنة (IACUC) من تكساس أية آند أم معهد "الطب الجينومي".
1-حل إعداد
<ol><li>تمييع حل هانك x 10 للملح متوازن إلى س 1 مع مزدوجة المقطر ح2س، وتخزينها في 4 درجات مئوية.</li>
	<li>إعداد الحل العزلة عن طريق إضافة هيبيس (10 ملم)، مجكل2 (1 ملم)، والسكر (5 ملم). ضبط ال pH إلى 7.4 والحفاظ على الجليد.</li>
	<li>إعداد الحل كولاجيناز من كولاجيناز نوع 4. ملاحظة: على وجه التحديد، كولاجيناز 4 نوع يحتوي على 160 ش كل مغ وزن الجاف. ز 1 من 4 نوع كولاجيناز هو المخفف في 5 مل مزدوجة المقطر ح2س بتركيز من 200 مغ/مل. 2 مل كولاجيناز المخفف يضاف إلى 30 مل الجليد الباردة من العزلة الحل، ويتم نقل 6 مل من محلول كولاجيناز إلى أنبوب 50 مل لكل الماوس.</li>
	<li>إعداد الحل أغسل بإضافة 1 مم كاكل2 إلى الحل العزلة.</li>
	<li>إعداد الحل تنقية مع الحل دياتريزواتي بوليسوكروسي الباردة/الصوديوم (1.1119 غ/مل، 5 مل).</li>
	<li>إعداد 500 مل من جزيرة الثقافة المتوسطة عن طريق إضافة لام الجلوتامين (20 ملم) والبنسلين (100 U/mL)، ستربتوميسين (100 ميكروغرام/مل) ومصل الدم البقري الجنين (FBS) (10%) في المتوسط ربمي.</li>
	<li>إعداد الحل دا دكفه الأسهم بتذويب المسحوق في ماء مقطر في 200 ميكرومتر ومخزن في-20 درجة مئوية. ملاحظة: روس تلطيخ الحل هو طازجة بتمييع الحل دا دكفه الأسهم إلى التركيز النهائي من 5 ميكرومتر في ربمي. وينبغي حماية هذا الكاشف من الضوء.</li>
</ol>2-العمليات الجراحية التحضير
<ol><li>حقن إينترابيريتونيلي (القائمة) ماوس مع أفيرتين (2.5% أفيرتين، 0.4 مل/20 g). بعد الماوس يتم تخديره كلياً ولم يعد يستجيب لهند القدم القرصات، euthanize الماوس ونقل إلى هود البيولوجية.</li>
	<li>ضع الماوس euthanized مع الجانب البطن مواجهة والرش الجلد مع الإيثانول 70% للتعقيم.</li>
	<li>فتح تجويف البطن مع يو-شق 1 سم على الجزء العلوي من البطن، وسحب الجلد في اتجاه روسترال على صدره لفضح تجويف البطن.</li>
</ol>3-البنكرياس نضح وإزالة
<ol><li>كما هو موضح في الشكل 1، ابحث عن موقع ampulla. استخدام زوج من منحنى ملقط المشبك العفج القرب من موقف المصرة العاصرة. استخدام زوج آخر من منحنى ملقط المشبك الجدار العفج إعاقة مسار الصفراوية في العفج. ملاحظة: لم تم إدراج في ampulla في هذه الخطوة. هذه الخطوة تهدف إلى عرقلة مسار الصفراوية في العفج.</li>
	<li>تغيير موضع الماوس مع الرأس الدانية والقدمين القاصي فيما يتعلق بالباحث.</li>
	<li>العثور على القناة الصفراوية المشتركة وحقن 3 مل من محلول كولاجيناز ببطء بحقنه إبرة ومل 5 ز 30. تأكد من القناة الصفراوية المشتركة والبنكرياس مبالغ فيها بعد الحقن.</li>
	<li>إزالة البنكرياس منتفخة باستخدام منحنى ملقط ومقص غرامة فصل تنازلي القولون والأمعاء والمعدة والطحال. ضع البنكرياس في أنبوب 50 مل يحتوي على 3 مل من محلول كولاجيناز وإبقائه على الجليد.</li>
</ol>4-البنكرياس الهضم
<ol><li>ضع كل أنبوب 50 مل في حمام مائي 37 درجة مئوية. احتضان لمدة 15-20 دقيقة (يتباين الوقت مع السلالة والعمر من الفئران).</li>
	<li>يهتز الأنبوبة باستخدام دوامة.</li>
	<li>تجمع 4-5 البنكرياس هضمها في مصفاة كبيرة بالوعة عقيمة واستخدم المجرفة جعل البنكرياس ينهار تماما. استخدام ز 23 إبرة و 10 مل حقنه تحتوي على الحل أغسل بقوة "الماصة؛" الخلايا الهضم خارج الشاشة. جمع الحل أغسل في أنبوب 50 مل.</li>
	<li>الطرد المركزي هذه الأنابيب في 97 x ز عند 4 درجة مئوية لمدة 1 دقيقة وصب المادة طافية.</li>
	<li>إضافة 25 مل من محلول الغسيل للأنابيب وإعادة تعليق الأنسجة التي فورتيكسينج لطيف. وتدور أسفل الأنسجة في 97 x ز عند 4 درجة مئوية لمدة 1 دقيقة صب المادة طافية. كرر 3 مرات. احرص على عدم إزاحة الأنسجة الأعلاف.</li>
</ol>5-تنقية للجزر الصغيرة
<ol><li>إضافة 10 مل من محلول تنقية لبيليه الأنسجة غسلها وريسوسبيند برفق المحتويات.</li>
	<li>تراكب 5 مل من محلول العزلة على الحل تنقية بلطف. كن حذراً للحفاظ على واجهة سائل حاد بين الحلين.</li>
	<li>الطرد المركزي الأنبوب في 560 × ز عند 4 درجة مئوية لمدة 15 دقيقة مع تسارع بطيء ولا الفرامل. إدراج الماصة في الواجهة وجمع طبقة الجزر الصغيرة في أنابيب 50 مل جديدة.</li>
	<li>أغسل الجزر الصغيرة كما هو موضح في الخطوة 3، 5.</li>
</ol>6-طلاء ومعاملة الخلايا جزيرة ليلى
<ol><li>ريسوسبيند الجزر الصغيرة في 10 مل من محلول الثقافة الجزيرات ولطف مزيج جيد ومكان 100 ميليلتر/جيدا مع ماصة متعددة القنوات لصفيحة 96-جيدا. ضع حوالي 5 الجزيرات الواحدة 96 جيدا.</li>
	<li>ضع اللوحة في الحاضنة زراعة الأنسجة ح 12.</li>
	<li>الطرد المركزي (112 س ز) لوحة 96-جيدا وإزالة الثقافة المتوسطة.</li>
	<li>تمييع المواد الكيميائية البيئية في الأجلين المتوسط وثقافة الجزر الصغيرة. إضافة 100 ميليلتر من كل من منظفة الكيميائية في آبار منفصلة من اللوحة. ريسوسبيند الجزر الصغيرة في كل بئر واحتضانها ح 12 في 37 درجة مئوية (يتغير التركيز المواد الكيميائية البيئية المختلفة).</li>
</ol>7-روس تلطيخ والقياس
<ol><li>بعد العلاج الجزر الصغيرة، وتغسل مع برنامج تلفزيوني 1 x (200 ميليلتر لكل بئر-96). ملاحظة: تركيز المواد الكيميائية على النحو التالي: AFB1: 3 ميكرومتر، والاترازين: 100 ميكرومتر، BaP: 10 ميكرومتر، منهاج عمل بيجين: 100 ميكرومتر، β-الإستروجين، PCB126: 10 ميكرون، وايثوكسيلات، ومنهاج عمل بيجين: 100 ميكرومتر.</li>
	<li>استخدام ن-أسيتيل سيستين (NAC) كمضاد للأكسدة لإخماد روس. إضافة 5 مم الائتلاف إلى منظفة الكيميائية الناجمة عن روس واحتضانها ح 12 عند 37 درجة مئوية.</li>
	<li>الطرد المركزي لوحة 96-جيدا في ز 112 x واحتضان مع 5 ميكرومتر الحل المسبار الفلورسنت (H2--دكفه--دا حله في وسائط الإعلام ربمي) عند 37 درجة مئوية لمدة 60 دقيقة.</li>
	<li>تغسل الخلايا المعالجة جزيرة ليلى 3 مرات مع برنامج تلفزيوني وقياس الأسفار مع قارئ ميكروسكوبية في 488 نانومتر. ملاحظة: ويمكن قياس تراكم المخصوم في الخلايا بزيادة في الأسفار في 530 نيوتن متر عند العينة هو متحمس في 488 نانومتر.</li>
	<li>تنفيذ المقايسة إفراز (نظام خدمات التأمين الحكومية) حفز الجلوكوز الأنسولين9 على إكسينوبيوتيك الذي يحفز روس تعامل الجزر والجزر الصغيرة غير المعالجة لقياس آثار إكسينوبيوتيك على الوظيفة الفسيولوجية جزيرات البنكرياس.</li>
</ol>

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الكتاب يعلن أن لديهم لا تضارب المصالح المالية.

تتراكم الأدلة تشير إلى أن التعرض للمواد الكيميائية البيئية دوراً هاما في تنمية T2DM. اعترف روس المستحثة xenobiotics عاملاً مسببا محتملة المساهمة في تنمية T2DM. ويتعرض البشر لمجموعة واسعة من المواد الكيميائية إكسينوبيوتيك وهناك حاجة كبيرة لتقنيات البحث رواية لتحديد فعالية سميات البنكرياس والتحقي...

أصبحت الزيادة في انتشار T2DM أزمة صحية عالمية في السنوات الأخيرة، مما يشكل تهديدا خطيرا للصحة العامة1. تم العثور على العديد من العوامل ارتباطاً سببياً تنمية T2DM، بينها، توحي النتائج المتكررة أن نقطة تلاقي مشتركة واحدة من هذه العوامل هو تنظيم دورات تعريفية للأكسدة مما يؤدي إلى توليد المفرط روس2 , 3.
تم العثور على مجموعة واسعة من المواد الكيميائية البيئية بما في ذلك ثنائي الفينيل متعدد الكلور والديوكسين و BaP للحث على الأكسدة، مما قد يضعف وظيفة خلايا بيتا في البنكرياس وتؤدي إلى مقاومة الأنسولين و T2DM4. على الرغم من أن مستوى الفسيولوجية روس دوراً هاما في الوظائف الخلوية، التعرض لروس الذي يتجاوز قدرة المنظومة المضادة للأكسدة ينتج الضرر لخلايا/الأنسجة ويؤدي إلى الأمراض5. الخلايا β البنكرياس التعبير عن مستوى منخفض من الإنزيمات المضادة للأكسدة، وهكذا هي هدف حساسة لأن الضرر عنصر مؤكسد الإجهاد بوساطة6،7. التعرض المزمن لمستويات عالية من روس قد ثبت أن تسبب الخلل في خلايا البنكرياس الناجمة عن الإجهاد5 فضلا عن مقاومة الأنسولين في الكبد والأنسجة الدهنية8.
الهدف العام لهذا المشروع تطوير تحليل يستند إلى الخلية للمواد الكيميائية الشاشة لإمكاناتها ديابيتوجينيك تستند على تحريض روس في خلايا البنكرياس. يفتقر إلى إزالة السموم الأيضية البنكرياس وهو هدف حساسة للأضرار التي يسببها إكسينوبيوتيك6،7. هذا التحليل بقياس روس المتولدة في الخلايا البنكرياس مباشرة، ولذلك ينبغي أن تقدم تقريب مباشرة من الضرر الناجم عن المواد الكيميائية في البنكرياس. لتطوير هذا الأسلوب، ونحن معزولة جزيرات البنكرياس الماوس ومثقف جزيرة معزولة تحت شرط الثقافة الخلية مع المواد الكيميائية وتستخدم الجيل روس الناجمة عن المواد الكيميائية كقراءات. ويتم هذا الإجراء البسيط والفعال في تحديد المواد الكيميائية الذي يحفز روس في جزيرة معزولة؛ فإنه يمكن تطوير لتحقيق آليات السمية الخاصة بالبنكرياس في المختبر.

<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

تم العثور على التعرض لبعض المواد الكيميائية البيئية في الإنسان والحيوان تسبب الأضرار الخلوية للخلايا β البنكرياس مما يؤدي إلى تطوير نوع 2 السكري (T2DM). على الرغم من أن آليات تلف الخلايا β الناجمة عن المواد الكيميائية غير واضح ومن المرجح أن تكون معقدة، إحدى النتائج المتكررة هي أن هذه المواد الكيميائية تحدث الأكسدة مما يؤدي إلى توليد الأنواع الأكسجين التفاعلية المفرط (روس) الذي حمل الأضرار التي لحقت خلية بيتا. لتحديد المواد الكيميائية البيئية المحتملة ديابيتوجينيك، نحن معزولة خلايا البنكرياس جزيرة ليلى من الفئران C57BL/6 وجزيرة مثقف الخلايا في خلية 96-جيدا لوحات الثقافة؛ ثم، كانت جرعتي الخلايا جزيرة ليلى مع المواد الكيميائية وتم الكشف عن توليد روس قبل 2 '7'-ديتشلوروفلوريسسين (دكفه-دا) صبغة الفلورسنت. باستخدام هذا الأسلوب، وجدت أن bisphenol A (BPA)، بنزو [أ] بيرين (BaP)، وثنائي الفينيل متعدد الكلور (PCBs)، يمكن أن تحفز مستويات عالية من روس، مما يوحي بأنها يحتمل أن تكون قد الحث على الأضرار في الخلايا من جزيرة ليلى. ينبغي أن يكون هذا الأسلوب مفيداً لفحص ديابيتوجينيك xenobiotics. وبالإضافة إلى ذلك، قد تكون الخلايا المستزرعة جزيرة ليلى أيضا تكييف للتحليل في المختبر للسمية الناجمة عن المواد الكيميائية في خلايا البنكرياس.

ويرد صورة مجهرية من جزيرة معزولة صحية في الشكل 2، التي يكون للجزر الصغيرة مستديرة أو بيضاوية شكل مع الزي الحجم نسبيا (على الرغم من أن حجم التوحيد يمكن أن تختلف من سلالة إلى سلالة). نحن التحقيق التالية وظائف البنكرياس جزيرة ليلى في فحص في المختبر بعزل ج?...

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

نقدم هنا بروتوكولا لعزل خلايا البنكرياس جزيرة الماوس لفحص عروض توضيحية روس قبل xenobiotics بغية تحديد المواد الكيميائية إكسينوبيوتيك ديابيتوجينيك المحتملة.

لفحص يستند إلى الخلية جزيرة البنكرياس مورين للمواد الكيميائية البيئية ديابيتوجينيك

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

Research

JoVE Journal

Immunology and Infection

7.2K Views.  Hunan Agriculture University. نقدم هنا بروتوكولا لعزل خلايا البنكرياس جزيرة الماوس لفحص عروض توضيحية روس قبل xenobiotics بغية تحديد المواد الكيميائية إكسينوبيوتيك ديابيتوجينيك المحتملة.

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.

فأري عزل جزيرة البنكرياس

Biology

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.

تحليلات ترانسكريبتوميك خلية واحدة من خلايا الغدد الصماء البنكرياس الماوس

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

Developmental Biology

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.

تحليل الرئيسيات غير البشرية جزيرة البنكرياس استهلاك الأكسجين

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; ديسيبل</sup&gt; نموذج الفأر من النوع 2 من مرض السكري: البنكرياس عزل جزيرة وتعيش خلية 2 فوتون التصوير من الجزر سليمة

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, ...

Medicine

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.

والمحتوى العالي<em&gt; في المختبر</em&gt; البنكرياس جزيرة β خلايا النسخ المتماثل منصة ديسكفري

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.

في Ovo نموذجية لاختبار مركبات الأنسولين ميميتيك

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

Neuroscience

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.

إينترابورتال زرع جزيرات البنكرياس في طراز الماوس

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.

وهناك طريقة للماوس البنكرياس جزيرة العزلة وبين الخلايا المخيم تقدير

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.

بروتوكول بسيط عالي الكفاءة لعزل الجزيرة البنكرياسية عن الفئران

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

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

تصوير ديناميات الكالسيوم في السكان الفرعيين من خلايا الفئران جزيرة البنكرياس

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

لفحص يستند إلى الخلية جزيرة البنكرياس مورين للمواد الكيميائية البيئية ديابيتوجينيك

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