نود أن نشكر رينيه L. Pasker وHarpreet K. برار للحصول على المساعدة التقنية للخبراء بشأن البروتوكولات وصفها في هذا العمل. وعلاوة على ذلك، نود أن نعترف التوجيه كريستوفر B. Newgard في جامعة ديوك وألان D. Attie في جامعة ويسكونسن ماديسون، جنبا إلى جنب مع دعم أعضاء المختبر، والتي سمح لنا الوقت والدعم اللازم لتحسين البروتوكولات وصفها. على وجه الخصوص، ونحن نشكر هانز Hohmeier، Danhong لو، وهيلينا وينفيلد في مختبر Newgard ومريم Rabaglia في مختبر Attie لمناقشات مثمرة والمشورة. وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح DK080845 وسكري الأطفال 594 منحة مؤسسة البحوث 17-2011-608 (لمجاهدي خلق)

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الكتاب ليس لديهم ما يكشف.

مع انتشار مرض السكري من المتوقع أن تؤثر على 7.7٪ من سكان العالم، واشتراط تقنيات البحث الرواية لا بد من فهم كل من وعلاج مرض السكري 18. عزلة جزيرة الحاضر هو بروتوكول راسخة المستخدمة في المختبر لالتجريب وقدمت من قبل مع تعديلات طفيفة 11،14،16. على الرغم من إفرا...

صيانة صارمة من سوائية سكر الدم أمر حتمي لمنع حالة مرضية مختلفة مثل اعتلال الأعصاب، اعتلال الكلية، واعتلال الشبكية، وهي كلها بصمات علم الأمراض من نوع غير المنضبط 1 و 2 من مرض السكري 1. انخفاض وظيفة الخلايا β والشامل في كل نوع 1 و 2 من مرض السكري التشويش على تركيز الجلوكوز في الدم 2. في حين أن نوع مناعية 1 نتائج مرض السكري من خسارة مدمرة لخلايا β-المنتجة للانسولين، وضعف إفراز الأنسولين خلايا β وإشارات الأنسولين الطرفية في مرض السكري من النوع 2 معا تعزيز ارتفاع السكر في الدم، دسليبيدميا، وزيادة إنتاج الجلوكوز في الكبد، مما يؤدي في نهاية المطاف في كلا فقدان القدرة كتلة خلايا β والانسولين إفرازية من فرد خلايا β 3. وفهم آليات خلايا β الكامنة في تطور النوع 2 من مرض السكري 1 و إعطاء أمل يؤدي إلى علاجات جديدة لمنع وعلاج هذه الأمراض. منظمة الشفافية الدولية في المختبرنماذج الثقافة ssue، مثل INS-1 وMIN6 خلد خطوط الخلايا β-، يمكن أن تكون أدوات مفيدة لفهم وظائف الخلايا β محددة. ومع ذلك، فإن التفاعلات بين أنواع مختلفة من الخلايا داخل جزيرة أنفسهم قد تنظم وظيفة الخلية β. على سبيل المثال، تأثير نظير الصماوي من الجلوكاجون (أفرج عنه من الخلايا α) والسوماتوستاتين (أفرج عنه من الخلايا δ) في زيادة وخفض إفراز الأنسولين، على التوالي، يدل على أهمية القرب خلية خلية في استجابة الغدد الصماء 4. علاوة على ذلك، تقاطعات الفجوة بين خلايا β تحفيز الافراج عن الانسولين 5. علاوة على ذلك، رغم ما اتخذ من خطوات في توليد خطوط ورم جزيري أن تكرارها بشكل أفضل الاستجابة الفسيولوجية من الجزر المعزولة إلى جلوكوز (على سبيل المثال، INS-1-مشتقة 832/13 و832/3 خطوط الخلايا)، والاستجابة الجلوكوز لا يزال يختلف من الفئران العادية الجزر 6،7. وعلاوة على ذلك، واستجابة لهذه خطوط الخلايا ورم جزيري النسيليلالببتيد-1 (GLP-1) منبهات مثل الجلوكاجون يمكن أن تختلف بشكل كبير عن بعضها البعض، وكذلك من الجزر الطبيعية 6. وبالتالي، خطوط الخلايا خلد قد لا تمثل أفضل نموذج لمعايرة العوامل التي تؤثر على إنتاج المخيم. وعلى النقيض من خطوط الخلايا المشتقة من ورم جزيري، ودراسة وظيفة خلايا β فقط في النماذج الحيوانية كلها تقدم مجموعتها الخاصة من المضاعفات. واحدة من أكبر التحديات في العمل مع أنسجة الغدد الصماء وقياس تركيز دقيق لهرمون يفرزه الجسم. على وجه التحديد، والكبد يلعب دورا رئيسيا تأييض الأنسولين، وتدفق الدم البنكرياس يذهب مباشرة إلى الكبد. وهكذا، فإن قياس الانسولين البلازما قد لا تصور بدقة كميات الأنسولين التي يفرزها البنكرياس من نفسه أو تأثير العلاجات المختلفة على معدل إفراز الأنسولين 8. وعلاوة على ذلك، والتمثيل الغذائي الكلوي من الجلوكاجون قد تحد من الاعتماد على الانتاج الجلوكاجون من خلايا جزيرة α 9. لذلك، عزل الجزر الماوس الأساسي للالتجريب في المختبر يوفر فهما أكثر دقة عن كيفية جزيرة يستجيب للمؤثرات محددة لاستكمال القياسات التي أجريت في الجسم الحي. هذا البروتوكول لعزل الجزر الماوس هو بروتوكول الراسخة التي يستخدمها عدد من المجموعات (مع بعض التعديلات الطفيفة التي قد تساعد على زيادة النجاح) 10،11. بالإضافة إلى ذلك، تحديد الإنتاج المخيم يسمح لقراءة المغادرة مباشرة للاستجابة incretin للخلايا β. بالتزامن مع قياس المخيم، ومحتوى البروتين إفراز الأنسولين كما يمكن كميا من نفس العينة المخيم الإعدادية، مما يساعد على تحديد ما إذا كان وجود خلل في وظيفة خلايا β يكمن الداني القاصي أو إلى معسكر 10. المحتوى المخيم النهائية وتطبيق إفراز الأنسولين في هذا البروتوكول يمكن أن يكون أداة قوية جدا لفهم تأثير المكونات الدوائية والغذائية، من بين أمور أخرىق، في المخيم وإفراز الأنسولين. بالإضافة إلى التحفيز من الجلوكوز وحده، ومركبات أخرى يمكن استخدامها لقياس التغيرات في المخيم وافراز الانسولين 10،11. أخيرا، على الرغم من أن الأنسولين هو الهرمون الأساسي أننا مقايسة من الجزر المعزولة، والهرمونات الأخرى، مثل الجلوكاجون والسوماتوستاتين، فضلا عن السيتوكينات، eicosanoids، وأحادي فسفات الأدينوزين الحلقي، ويمكن أيضا أن تقاس، إما عن طريق تحفيز مقايسة عابرة أو عن طريق التحديد الكمي لل مستوياتها في مستنبت 12. أخيرا، على الرغم من خارج نطاق هذه المخطوطة، والعزلة جزيرة مع أسلوب كولاجيناز العزلة وصفها يسمح للحفاظ على جزيرة بحيث يمكن السعي العديد من التطبيقات المصب الأخرى، مثل زرع جزيرة، عزل الحمض النووي الريبي في الوقت الحقيقي PCR الكمي أو ميكروأري التحليلات، البروتين العزلة عن النشاف الغربية، جزيرة التضمين والتصوير مناعي، وإدماج [3H]-ثيميدين كمقياس للrepli خلايا الجزيرةالموجبة، وبعضها قد تم وصفها في مقالات سابقة إن الرب 13-16. عموما، بعد إجراء العزل جزيرة موضح في البروتوكول قد توفر الباحث بمعلومات هامة ومفيدة لتطوير علاجات وتشجيع اكتشاف المخدرات التي تهدف إلى تعزيز وظيفة خلايا β.

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

سكر الدم غير المنضبط هو السمة المميزة لمرض السكري ويعزز حالة مرضية مختلفة مثل اعتلال الأعصاب، اعتلال الكلية، واعتلال الشبكية. مع الانتشار المتزايد لمرض السكري، وكلاهما مناعية من نوع 1 ونوع 2 المرتبط بالسمنة، والدراسات التي تهدف إلى ترسيم الفيزيولوجيا المرضية السكري والآليات العلاجية هي ذات أهمية حاسمة. الخلايا β لجزر لانجرهانز في البنكرياس المسؤولة عن إفراز الأنسولين بشكل مناسب في استجابة لتركيزات السكر في الدم مرتفعة. بالإضافة إلى الجلوكوز والمواد المغذية الأخرى، يتم تحفيز خلايا β أيضا هرمونات محددة، ووصف incretins، والتي تفرز من الأمعاء ردا على وجبة والعمل على مستقبلات خلايا β التي تزيد من إنتاج الخلايا أحادي فسفات الأدينوزين الحلقي ( المخيم). انخفاض وظيفة الخلايا β-، الشامل، والاستجابة incretin هي مفهومة جيدا للمساهمة في الفيزيولوجيا المرضية لمرض السكري من النوع 2، ويجري أيضا مرتبطة على نحو متزايد وايال داء السكري نوع 1. الماوس الحاضر جزيرة العزلة ومخيم بروتوكول تقرير يمكن أن يكون أداة للمساعدة في ترسيم آليات تعزيز تطور المرض والتدخلات العلاجية، وخاصة تلك التي يتم بوساطة المستقبلات incretin أو المستقبلات التي تعمل من خلال تعديل الإنتاج المخيم الخلايا ذات الصلة. في حين سيتم وصفها القياسات المخيم فقط، بروتوكول جزيرة العزلة وصفها يخلق إعداد النظيفة التي تسمح أيضا للعديد من التطبيقات المصب الأخرى، بما في ذلك الجلوكوز حفز افراز الانسولين، [3 H] ثيميدين-التأسيس، وفرة البروتين، والتعبير مرنا.

لضمان العائد جزيرة عالية خلال العزلة، ينبغي اتباع التقنيات الجراحية المذكورة في البروتوكول عن كثب. على الرغم من أن التقنيات المعروضة هنا سوف يكون متلائما مع كل مختبر، وهناك عدد قليل من الخطوات الحاسمة التي من شأنها أن تؤدي إلى العزلة الناجحة. من أجل جعل القناة الصفر?...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

يعاير في المختبر وظيفة β-الخلية باستخدام الماوس الجزر المعزولة لانجرهانز هو عنصر هام في دراسة الفيزيولوجيا المرضية السكري والعلاجات. في حين أن العديد من التطبيقات المتاحة المصب، ويصف هذا البروتوكول على وجه التحديد قياس الخلايا أحادي فسفات الأدينوزين الحلقي (المخيم) كمعلمة أساسية تحديد وظيفة خلايا β.

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 ...

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Research

JoVE Journal

Biology

65.0K Views.  University of Wisconsin-Madison.  يعاير في المختبر وظيفة β-الخلية باستخدام الماوس الجزر المعزولة لانجرهانز هو عنصر هام في دراسة الفيزيولوجيا المرضية السكري والعلاجات. في حين أن العديد من التطبيقات المتاحة المصب، ويصف هذا البروتوكول على وجه التحديد قياس الخلايا أحاد?...

Video: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Murine Pancreatic Islet Isolation

فأري عزل جزيرة البنكرياس

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.

الإنسان عزل جزيرة البنكرياس : الجزء الأول : الهضم ومجموعة من الأنسجة البنكرياس

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

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

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of 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 characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we 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. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

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 II: purification and culture of human islets) using a modified automated method.

الإنسان عزل جزيرة البنكرياس : الجزء الثاني : تنقية الثقافة والإنسان من الفشوت

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

بمساعدة الحاسوب على نطاق واسع التصور النوعي والكمي لقداس جزيرة البنكرياس والتوزيع الحجم والعمارة

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

الماوس جزيرة من العزلة انجرهانز باستخدام مزيج من كولاجيناز تنقية والبروتياز متعادلة

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

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.

العزلة والثقافة من الماوس الأساسي خلايا البنكرياس عنيبية

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 ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

بالقرب الأشعة تحت الحمراء (نير) ضوء يزيد من التعبير عن علامة من الميتوكوندريا وظيفة في ماوس الدهليزي الحسية الظهارة

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

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 ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

طريقة لزرع جزيرة ليلى إلى الثرب في الماوس

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

جزيرة البنكرياس التضمين لمقاطع البارافين

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...