ويدعم هذا العمل من المنح المقدمة من الصحة الوطنية الاسترالية ومجلس البحوث الطبية (NHMRC # 559007) والأحداث السكري مؤسسة أبحاث (JDRF 4-2006-1025) ومخطط البنية التحتية التشغيلية لحكومة فيكتوريا. نشكر أعضاء ماندل توم برنامج زرع جزيرة عزل فريق جزيرة لتوفير الأنسجة البشرية. تم جمع الأنسجة البشرية واستخدامها مع الموافقة الأخلاقية المحلية (مستشفى سانت فنسنت HREC-A 011/04 والصحة سانت فنسنت HREC-A 135/08).

1. إعداد الأنسجة الطحال <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ينبغي أن يعامل الملاحظات جميع المواد التي قد تكون معدية الإنسان وينبغي أن تجري جميع الإجراءات في مجلس الوزراء من الفئة الثانية تدفق الصفحي. باستخدام مقص ملقط معقم و، وإزالة الأنسجة الدهنية والليفية من أقسام الطحال (~ 1-2 سم في حجم) وتقليم قبالة أكبر قدر من المواد كبسولة الخارجي قدر الإمكان. </li><li style=";text-align:right;direction:rtl"> قطع قطعة صغيرة (1-2 سم 3) من الأنسجة الطحال ووضع كل قطعة في أنبوب العقيمة 50 مل فالكون. </li><li style=";text-align:right;direction:rtl"> الأداة الإضافية تجميد قطعة من الأنسجة عن طريق الغمر في السائل N 2. </li><li style=";text-align:right;direction:rtl"> المحل في -80 درجة مئوية. بروتوكول مماثلة مناسبة لالأنسجة الأخرى (ق). </li></ol> 2. إعداد الإنسان للجزيرة التخزين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الجزر الثقافة في وسائل الإعلام CMRL. جمع الجزر في أنبوب 10 مل المخروطية وغسل أسفل مرتين في برنامج تلفزيوني بواسطة الطرد المركزي في 1،500 دورة في الدقيقة لمدة 5 دقائق. صب قبالة PBS واستنزاف المخزن المؤقت المتبقية عن طريق وضع أنبوب مقلوب لفترة وجيزة على منشفة ورقية. يجب الحرص على عدملإزاحة الجزر. </li><li style=";text-align:right;direction:rtl"> استنزفت مرة واحدة، وإعادة رسملة الأنبوب والمفاجئة التجميد في النيتروجين السائل وتخزينها في -80 درجة مئوية. </li></ol> 3. إعداد مستخلصات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد مزيج BAW (10:30:60٪ V / V) وتخزينها في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة الأنبوب من C. ° -80 ذوبان الجليد في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إضافة ما يكفي من الجليد الباردة BAW لتغطية قطعة من نسيج الجسم. لاستخدام الجزر 3-5 مل. لاستخدام الأنسجة الطحال 10-20 مل تبعا لحجم قطعة من نسيج الجسم. </li><li style=";text-align:right;direction:rtl"> تجميع الخالط الأنسجة. تنظيف ل &#39;المجانسة &quot;10-20 مل من الايثانول 70٪ / المياه. </li><li style=";text-align:right;direction:rtl"> تجانس الأنسجة في رشقات نارية عدة، بعد وضع التحقيق في أنبوب الخالط مع الأنسجة وحل BAW. إبقاء الأنبوب في دلو الجليد بعد ذلك. </li><li style=";text-align:right;direction:rtl"> تنظيف بدقة الخالط بين العينات، من خلال التجانس 10-20 مل من الايثانول 70٪ / الماء ثم العازلة BAW. هذا يمنع انتقال التلوث من الأنسجة بين samples.Dismantleand نظيفة مع الايثانول 70٪ / من بعدنا المياهه. </li><li style=";text-align:right;direction:rtl"> إذا كان المطلوب مقتطف تحتوي على مواد قابلة للذوبان فقط، المتجانس أجهزة الطرد المركزي في استخراج أنسجة 4،000 دورة في الدقيقة في RT لمدة 10 دقيقة. إذا كنت بحاجة لاستخراج مزيد من النفط الخام، وتدور في 1،000 دورة في الدقيقة في RT لمدة 5 دقائق. الأسلوب الأكثر ملائمة لاستخراج البروتينات غير قابلة للذوبان BAW يعتمد على تحليل المصب من البروتينات. لاستخدامها في المقايسات المناعية الوظيفية نقترح محاولة حل جزء غير قابل للذوبان في اليوريا M 8 إذ أننا وجدنا في السابق أن هذا جيد التحمل 6. </li><li style=";text-align:right;direction:rtl"> نقل طاف لأنبوب نظيفة ووضعها على الجليد. </li><li style=";text-align:right;direction:rtl"> تحديد تركيز البروتين في استخراج باستخدام مقايسة BCA أو ما شابه ذلك. </li></ol> 4. تجميد التجفيف مقتطفات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اعتمادا على كتلة المطلوبة من البروتين (أي 100 ميكروغرام لكل أنبوب)، وفقا لتخفيف جناسة والاستغناء مأخوذة في المسمى عقيمة مل 5.0، الصقر (12x75 ملم) الأنابيب. ونحن كثيرا ما تستخدم 100 ميكروغرام / الأنبوب. </li><li style=";text-align:right;direction:rtl"> استخدام 18-20 إبرة قياس محقنة معقمة تقديم 3 ثقوب في سقف كل أنبوب. </li><li style=";text-align:right;direction:rtl"> تجميد أنابيب إما عن طريق وضعها على الثلج الجاف ل10 دقيقة ~ أو في الفريزر -80 ° C للساعة 1&gt;. المحل في -80 درجة مئوية حتى على استعداد لوضع على مجفاد. </li><li style=";text-align:right;direction:rtl"> تشغيل أكثر جفافا للتجمد والسماح للتوازن (-100 درجة مئوية). هذا يستغرق حوالي 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> وهذا يتوقف على حجم، ويمكن الانتهاء تجميد التجفيف بعد 3 ساعة ولكن نترك بشكل روتيني عينات لدينا ليلة وضحاها. </li><li style=";text-align:right;direction:rtl"> بعد الانتهاء من دورة التجفيف، وإيقاف مضخة فراغ، والسماح ببطء الى غرفة الضغط. إزالة الحامل. </li><li style=";text-align:right;direction:rtl"> في غطاء محرك السيارة العقيمة، تنزع الأغطية من أنابيب مثقبة واستبدالها بأخرى جديدة (فالكون 352032). </li><li style=";text-align:right;direction:rtl"> أنابيب مخزن في -20 ° C. يمكن تشكيل العينات في وسائل الإعلام والثقافة، وأو مخازن أخرى، وتستخدم في وظيفة أو المقايسات الكيميائية الحيوية. </li></ol> 5. ممثل النتائج ويظهر الشكل 1 تلطيخ من البروتينتحميل هلام مع مقتطف من الطحال والبنكرياس جزيرة الأنسجة المنضب (المسمى عنيبية) وتنقيته الجزر الإنسان (الجزر المسماة). النتائج تظهر تمثيل جيد للبروتينات ذات الوزن الجزيئي من مختلف لكل الأنسجة. تم اختبار قدرة مقتطفات الأنسجة البشرية لتحفيز تكاثر الخلايا T-باستخدام مقايسة انتشار CFSE القائم 7 (الشكل 2). تم عزل PBMC المستخدمة في هذا الاختبار من أي فرد يعانون من مرض السكري من النوع 1. يتم التعبير عن حجم الاستجابة كنسبة من عدد الخلايا CFSE قاتمة في 5000 CD4 + الخلايا CFSE، ومشرق دون مستضد: من الخلايا CFSE قاتمة في 5000 CD4 +، وخلايا CFSE مشرقة مع مستضد ثلاث نسخ من عينات 7. النتائج تظهر ضعيفة، ولكن يمكن اكتشافها، والانتشار ردا على عنيبية (CD1 = 3.5) وأقوى استجابة لاستخراج جزيرة (6.8). يتم تضمين فيروس الأنفلونزا المعطل (CDI = 142.6) وذلكإيجابية السيطرة. <img alt="الشكل 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> الشكل 1. هلام بروتين. <img alt="الشكل 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> الشكل 2. نتائج الفحص من انتشار CFSE القائم ضد عنيبية واستخراج جزيرة. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">اضغط هنا لمشاهدتها بشكل اكبر شخصية</a> .

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	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

الإعلان عن أي تضارب في المصالح.

وقد تم تطوير هذا البروتوكول لأننا أردنا لتوليد مقتطف من الأنسجة البشرية التي كانت خالية من المواد الكيميائية السامة مثل المنظفات. على وجه التحديد وقد استخدمنا ذلك لإعداد مقتطفات من الأنسجة البشرية التي يمكن استخدامها في فحوصات وظائف المناعة البشرية في المختبر.</em...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> 5 مل 12 × 75 مم أنابيب معقمة البوليسترين </td><td> BD الصقر </td><td> 352054 </td><td></td></tr><tr><td> قبعات للأنابيب أنابيب البولي ستايرين (أعلاه) </td><td> BD الصقر </td><td> 352032 </td><td></td></tr><tr><td> 50ML أنابيب معقمة </td><td> بيكتون ديكنسون </td><td> 352070 </td><td></td></tr><tr><td> الأسيتونيتريل </td><td> كيماويات Mallinckradt </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-OL </td><td> سيغما الدريتش </td><td> 537993-IL </td><td></td></tr><tr><td> الخالط: PRO200 </td><td> الحيوية الاستراتيجية </td><td> 01-01200 </td><td> 10 × 115 مم المنشار الأسنان مولد </td></tr><tr><td> مجفاد </td><td></td><td></td><td> Virtis، الفوق 4K </td></tr><tr><td> إبرة معقمة قياس 18-20 </td><td> بيكتون ديكنسون </td><td> REF 302032 </td><td></td></tr><tr><td> CMRL-1066 متوسطة </td><td> سيجما </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> سيجما </td><td> D8537 </td><td></td></tr></tbody></table> الجدول 1. الكواشف محددة والمعدات.

extraction of tissue antigens for functional assays

العديد من الأهداف مستضد من الاستجابة المناعية التكيفية، معترف بها من قبل والخلايا B T، لم تعرف 1. هذا صحيح بصفة خاصة في أمراض المناعة الذاتية والسرطان 2. هدفنا هو التحقيق في مستضدات الخلايا T التي تعترف بها الإنسان في نوع 1 من داء السكري أمراض المناعة الذاتية 1،3،4،5. لتحليل خلايا T الإنسان استجابات ضد الأنسجة حيث لا تحديد مستضدات الخلايا T معترف بها من قبل وضعنا طريقة لاستخراج البروتين من مولدات المضادات الأنسجة البشرية في تنسيق متوافق مع المقايسات الفنية 6. لا يمكن أن الردود سابقا، T-خلية الأنسجة غير منقى لمقتطفات أن تقاس لأن طرق استخراج تسفر عن المحللة التي تحتوي المنظفات التي كانت سامة للخلايا الدموية المحيطية وحيدات النوى. نحن هنا وصف بروتوكول لاستخراج البروتينات من الأنسجة البشرية في شكل غير سامة للخلايا T الإنسان. والمتجانس النسيج في خليط من الأسيتونيتريل، butan-1-ol و واتإيه (BAW). يتم قياس تركيز البروتين في استخراج أنسجة وaliquoted كتلة معروفة من البروتين في أنابيب. بعد الاستخراج، يتم إزالة المذيبات العضوية من قبل تجفيد. ويمكن تخزين مقتطفات الأنسجة مجفف بالتجميد حتى المطلوبة. لاستخدامها في فحوصات وظائف المناعة، وتعليق من الخلايا المناعية، في وسائل الإعلام الملائمة الثقافة، يمكن إضافة مباشرة إلى استخراج مجفف بالتجميد. تم قياس بسهولة وانتشار إنتاج السيتوكينات بواسطة PBMC، ردا على مقتطفات إعدادها باستخدام هذا الأسلوب. وبالتالي، لدينا أسلوب يسمح للإعداد السريع للأنسجة البشرية لست] التي يمكن استخدامها كمصدر للمستضدات في تحليل الخلايا T-الردود. نقترح أن هذا الأسلوب سيسهل تحليل الاستجابات المناعية التكيفية في زرع الأنسجة لوالسرطان والمناعة الذاتية.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

يوصف بروتوكول بسيط لإعداد مقتطفات من الأنسجة البشرية لاستخدامها كمصدر للمستضدات في المقايسات T-الخلية الوظيفية. هذا الأسلوب يسمح للقياس T-الخلية الردود على الأنسجة المشتقة من المستضدات<em&gt; في المختبر</em&gt;.

استخراج المستضدات لفحوصات الأنسجة وظيفية

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

extraction-of-tissue-antigens-for-functional-assays

Research

JoVE Journal

Biology

10.8K Views.  St. Vincent's Institute of Medical Research. يوصف بروتوكول بسيط لإعداد مقتطفات من الأنسجة البشرية لاستخدامها كمصدر للمستضدات في المقايسات T-الخلية الوظيفية. هذا الأسلوب يسمح للقياس T-الخلية الردود على الأنسجة المشتقة من المستضدات في المختبر.

Video: Extraction of Tissue Antigens for Functional Assays

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

الضوء / الظلام لاختبار الانتقال الفئران

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

أكتين المشارك الترسيب الفحص ، وبالنسبة للتحليل البروتين ملزمة لF - أكتين

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

بلورة البروتينات الغشائية لتحديد الهيكل باستخدام Mesophases Lipidic

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

فحوصات الوراثية الدوائية وظيفية للتلاعب تجديد مستورقة Dugesia تفرضه اليابان</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

الكمية مقارنة رابطة الدول المستقلة التنظيم العنصر (لجنة المساواة العرقية) في الأنشطة المعدلة وراثيا ذبابة الفاكهة السوداء البطن</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

استخدام روبوت لارتفاع الإنتاجية البلورة من بروتينات الغشاء في Mesophases شحمي

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

على نطاق صغير مقتطفات النووية لفحوصات وظيفي من الجينات التعبير عن مكائن

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

فحوصات قابلية للخلايا في الثقافة

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

العزلة والتحليل الوظيفي من الميتوكوندريا من الخلايا المستزرعة وماوس الأنسجة

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

إعداد النوى الأنسجة الثابتة الفورمالين جزءا لا يتجزأ من البارافين لكلا RNA واستخراج الحمض النووي

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...