وأيد هذه الدراسة من قبل القومي للقلب والرئة والدم المعهد جرانت HL- 103542، وجائزة تريل بليزر المتميزة المؤسسة الغرفة بحوث التخدير لCAP.

إعداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 1 M-K HEPES محلول المخزون (ضبط درجة الحموضة إلى 7.2 مع KOH). </li><li style=";text-align:right;direction:rtl"> تحضير 0.5 M-K EGTA المالية الحل (ضبط درجة الحموضة إلى 8.0 مع KOH). </li><li style=";text-align:right;direction:rtl"> إعداد 1 M KH 2 PO 4 أسهم الحل. </li><li style=";text-align:right;direction:rtl"> إعداد 1 M MgCl 2 محلول المخزون. </li><li style=";text-align:right;direction:rtl"> إعداد المجانسة العازلة (درجة الحموضة 7.2) 300 ملي السكروز، K-10 ملي HEPES، وK-1 ملم EGTA. تخزينها في 4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إعداد التنفس العازلة 250 ملي السكروز، 2 مم KH 2 PO 4، 10 ملم MgCl 2 و 20 ملي K-HEPES العازلة (درجة الحموضة 7.2) و 0.5 ملي K-EGTA (8.0 درجة الحموضة). تخزينها في 4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إعداد 10X PBS المالية الحل عن طريق إذابة 80 جم من كلوريد الصوديوم، و 2 غرام من بوكل، 14.4 غرام من نا 2 هبو 4، و 2.4 غرام من KH 2 PO 4 في 1 L مزدوج المقطر H 2 O (7.4 درجة الحموضة). </li><li style=";text-align:right;direction:rtl"> إعداد 1X PBS قبل pipetting 100 مل 10X PBS إلى 1 L مزدوج المقطر H 2 O. </li><li style=";text-align:right;direction:rtl"> إعداد سبتيليزين مخزون من وزنها من 4 ملغ من سبتيليزين ألف فيإلى أنبوب 1.5 مل microfuge. تخزين في -20 درجة مئوية حتى الاستخدام. </li><li style=";text-align:right;direction:rtl"> إعداد BSA المالية التي وزنها من 20 ملغ من BSA في أنبوب 1.5 مل microfuge. تخزين في -20 درجة مئوية حتى الاستخدام. </li></ol> عزل الميتوكوندريا (الشكل 1) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مباشرة قبل العزلة، ويحل سبتيليزين ألف في 1 مل من المجانسة العازلة. </li><li style=";text-align:right;direction:rtl"> مباشرة قبل العزلة، ويحل BSA في 1 مل من المجانسة العازلة. </li><li style=";text-align:right;direction:rtl"> جمع عينتين أنسجة جديدة باستخدام 6 ملم عينة خزعة لكمة وتخزينها في برنامج تلفزيوني 1X في أنبوب الطرد المركزي 50 مل مخروطي على الجليد. </li><li style=";text-align:right;direction:rtl"> نقل اثنين من اللكمات 6 ملم من الأنسجة لأنبوب التفكك C تحتوي على 5 مل من البرد الجليد التجانس العازلة. </li><li style=";text-align:right;direction:rtl"> تجانس الأنسجة عن طريق تركيب أنبوب C التفكك على dissociator الأنسجة وتحديد دورة العزلة الميتوكوندريا المحددة مسبقا (60 ثانية التجانس). </li><li style=";text-align:right;direction:rtl"> إزالة أنبوب التفكك C إلى دلو الجليد. </li><li style=";text-align:right;direction:rtl"> إضافة 250 ميكرولتر من سبتيليزين مخزون الحل لجناسة، مزيج من انعكاس واحتضان جناسة على الجليد لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> وضع مرشح شبكة 40 ميكرون على أنبوب الطرد المركزي 50 مل مخروطي على الجليد وما قبل الرطب مرشح مع المجانسة العازلة وتصفية جناسة في أنبوب الطرد المركزي 50 مل مخروطي على الجليد. </li><li style=";text-align:right;direction:rtl"> إضافة 250 ميكرولتر من الطازجة BSA الحل لاسهم الراشح وتخلط بواسطة انقلاب. ملاحظة: حذفت هذه الخطوة إذا كان مطلوبا الميتوكوندريا تقرير البروتين. </li><li style=";text-align:right;direction:rtl"> وضع مرشح 40 ميكرون على أنبوب الطرد المركزي 50 مل مخروطي على الجليد وما قبل الرطب مرشح مع المجانسة العازلة وتصفية جناسة في أنبوب الطرد المركزي 50 مل مخروطي على الجليد. </li><li style=";text-align:right;direction:rtl"> وضع مرشح 10 ميكرون على أنبوب الطرد المركزي 50 مل مخروطي على الجليد وما قبل الرطب مرشح مع المجانسة العازلة وتصفية جناسة في أنبوب الطرد المركزي 50 مل مخروطي على الجليد. </li><li style=";text-align:right;direction:rtl"> نقل الترشيح لاثنين قبل المبردة 1.5 مل أنابيب microfuge والطرد المركزي في 9000 x ج لمدة 10 دقيقة في4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إزالة طاف وإعادة تعليق والجمع بين الكريات في 1 مل من الجليد الباردة التنفس العازلة. </li></ol> ATP الفحص ملاحظة: لتحديد النشاط الأيضي الميتوكوندريا المعزولة والتلألؤ فحص ATP يمكن أن يؤديها باستخدام عدة ATP الفحص. بروتوكول، تم تزويد الكواشف والمعايير في عدة الفحص. يوصف ملخص الإجراء أدناه. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تتوازن الكواشف عدة لRT. </li><li style=";text-align:right;direction:rtl"> إعداد 10 ملي ATP المالية الحل عن طريق إذابة بيليه ATP مجفف بالتجميد في 1،170 ميكرولتر من الماء المقطر مزدوجة. تخزين ATP المالية القياسية الحل وعينات الميتوكوندريا مستعدة على الجليد. </li><li style=";text-align:right;direction:rtl"> إضافة 5 مل من محلول الركيزة العازلة إلى قارورة من حل الركيزة مجفف بالتجميد. المزيج بلطف ووضع في الظلام. </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من العازلة التنفس لجميع آبار سوداء، أسفل مبهمة، 96 لوحة جيدا. </li><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من الميتوكوندريا من العينات مستعدة لكل بئر ساتحاد كرة القدم الأسود، أسفل مبهمة، 96 لوحة جيدا. ملاحظة: يتم مطلي العينات في ثلاث نسخ. تشمل على التوالي لمعايير وثلاثة آبار للسيطرة سلبية (التنفس الاحتياطي). </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من الثدييات حل تحلل الخلية لجميع الآبار، بما في ذلك المعايير والضوابط. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة عند 37 درجة مئوية لمدة 5 دقائق على شاكر المداري في 125 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> أثناء الحضانة إعداد معايير ATP في تركيزات 0.1 ملم، 0.05 ملم، 0.01 ملم، 0.005 ملم، 0.001 ملم، و0.0001 ملي ATP من 10 ملي ATP المالية الحل. مخزن المعايير على الجليد. </li><li style=";text-align:right;direction:rtl"> بعد الحضانة، وإضافة 10 ميكرولتر من المعايير ATP إلى الآبار المقابلة كما هو موضح على الخريطة وحة. يتم تنفيذ المعايير في نسختين: مذكرة. </li><li style=";text-align:right;direction:rtl"> إضافة 50 ميكرولتر من الحل الركيزة أعيد إلى كل بئر. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة عند 37 درجة مئوية على شاكر المداري لمدة 5 دقائق عند 125 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> مفتوحة Gen5 1.11 البرنامج على جهاز كمبيوتر مرتبط مع طيفي. </li><li style=";text-align:right;direction:rtl"> تحت عنوان &quot;إنشاء عنصر جديد &quot;، انقر على&quot; تجربة &quot;. </li><li style=";text-align:right;direction:rtl"> انقر على &quot;بروتوكول الافتراضي&quot;. انقر على &quot;موافق&quot;. </li><li style=";text-align:right;direction:rtl"> في العمود الأيسر، حدد &quot;البروتوكول&quot;، ثم &quot;الداخلي&quot;. </li><li style=";text-align:right;direction:rtl"> حدد &quot;تأخير&quot;. تعيين ل00:10:00. انقر على &quot;موافق&quot;. </li><li style=";text-align:right;direction:rtl"> حدد &quot;اقرأ&quot;. حدد &quot;التلألؤ&quot; من القائمة المنسدلة. ضبط الإعدادات الأخرى إلى ما يلي: اقرأ نوع نقطة النهاية والتكامل الوقت 0: 01.00 MM: SS.ss، تصفية مجموعات 1، الانبعاثات هول، البصريات الوظيفة الأعلى، الحساسية 100، أوفست الأعلى المسبار الرأسي 1.00 ملم. </li><li style=";text-align:right;direction:rtl"> عندما لوحة جاهزة للتحليل، انقر فوق &quot;قراءة لوحة&quot; أيقونة على الصف العلوي. انقر على &quot;اقرأ&quot;. عندما يفتح طيفي، ووضع لوحة في علبة مع جيدا A1 في الزاوية اليسرى العليا. سوف يظهر مربع مع درجة حرارة فتح. انقر على &quot;اقرأ&quot;. ملاحظة: القيم العليا ترتبط مع زيادة مستويات ATP والنشاط الأيضي العالي. </li></ol>

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تم علاج جميع الحيوانات وفقا للمبادئ التوجيهية المؤسسية الحالية. ليس لدينا مصالح المالية المتنافسة في الكشف عنها.

لعزل الميتوكوندريا بنجاح باستخدام هذا البروتوكول لا بد من الحفاظ على كل الحلول وعينات الأنسجة على الجليد للحفاظ على استمرارية الميتوكوندريا. حتى عندما حافظت على الجليد، والميتوكوندريا المعزولة تظهر انخفاضا في النشاط الوظيفي على مر الزمن 19. ونحن نوصي جميع الح...

A correction was made to: <a href="http://www.jove.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

Erratum: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

وتوجد الميتوكوندريا في كل خلية في الجسم ما عدا خلايا الدم الحمراء وتشارك في عدد كبير من العمليات الخلوية والأيض المهمة 1-4. لأن العديد من هذه الوظائف، يمكن أن تلف الميتوكوندريا لها آثار ضارة 3. من أجل التحقيق في وظيفة الميتوكوندريا وضعف عدة طرق العزلة الميتوكوندريا تم وصفها. أقرب الحسابات المنشورة التاريخ العزلة الميتوكوندريا إلى 1940s 5-8. أظهرت محاولة موثقة الأولى العزلة الميتوكوندريا عن طريق طحن أنسجة الكبد في بمدافع الهاون تليها الطرد المركزي في محلول الملح على سرعة منخفضة 5،8. في وقت لاحق، وسعت مجموعات أخرى على الإجراء الأصلي وأظهرت الأنسجة تجزئة استنادا التفاضلية الطرد المركزي 6-8. هذه الأساليب المبكرة شكلت أساس التقنيات الحالية والتي غالبا ما تتضمن التجانس، و / أو التفاضلية الطرد المركزي 9-15. عدد homogenizatiعلى الطرد المركزي والخطوات يختلف بين البروتوكولات. هذه الخطوات المتكررة تزيد من الوقت لعزل الميتوكوندريا وفي نهاية المطاف خفض قدرتها على البقاء. بالإضافة إلى ذلك، يمكن أن تسبب أضرارا التجانس اليدوي وتتعارض النتائج الميتوكوندريا إذا لم يتم السيطرة بشكل صحيح 10،16. في الآونة الأخيرة، كنا التجانس والتفضيلية الطرد المركزي لعزل الميتوكوندريا لزرعها في أنسجة عضلة القلب 17،18. هذا الإجراء يتطلب عزلة طويلة ما يقرب من 90 دقيقة وكان انطباق السريري لهذه الطريقة بالتالي محدود. للسماح للاستخدام العلاجي الحاد في العلاج السريري والجراحية وضعنا الداخلي العزلة الميتوكوندريا السريع التي يمكن القيام بها في أقل من 30 دقيقة. الفوائد الرئيسية لهذا البروتوكول هي أن تفكك نسيج موحد يسمح موحدة وتجانس ثابت من الأنسجة التي لا يتحقق بسهولة مع التجانس اليدوي. في additأيون، واستخدام الترشيح التفاضلي في مكان الطرد المركزي التفاضلي يلغي تستغرق وقتا طويلا والخطوات الطرد المركزي المتكررة السماح لعزل أسرع من الميتوكوندريا المختصة العالية النقاء وقابلة للحياة والتنفس. القدرة على عزل الميتوكوندريا المختصة قابلة للحياة والتنفس في أقل من 30 دقيقة تسمح للتطبيق السريري. هذا البروتوكول العزلة لديها امكانات للاستخدام في جراحة الشريان التاجي الالتفافية تطعيم (CABG) والإجراءات العلاجية الأخرى.

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rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

طرق العزلة الميتوكوندريا التي سبق وصفها باستخدام الطرد المركزي التفاضلي و / أو Ficoll التدرج الطرد المركزي تتطلب 60 الى 100 دقيقة لإكمال. نحن تصف طريقة لعزل الميتوكوندريا السريع لخزعات من الثدييات باستخدام dissociator النسيج التجاري والترشيح التفاضلي. في هذا البروتوكول، يتم استبدال التجانس اليدوي مع دورة التجانس موحدة للdissociator في الأنسجة. وهذا يسمح للموحدة وتجانس ثابت من الأنسجة التي لا يتحقق بسهولة مع التجانس اليدوي. بعد تفكك الأنسجة، ويتم تصفية جناسة من خلال شبكة مرشحات النايلون، والتي تزيل الخطوات الطرد المركزي المتكررة. ونتيجة لذلك، والعزلة الميتوكوندريا يمكن أن يؤديها في أقل من 30 دقيقة. هذا البروتوكول العزلة ينتج حوالي 2 × 10 10 الميتوكوندريا المختصة قابلة للحياة والتنفس من 0.18 ± 0.04 جرام (الوزن الرطب) عينة الأنسجة.

ويرد الرقم يحدد الخطوات الإجرائية في عزل الميتوكوندريا باستخدام تفكك الأنسجة والترشيح الفارق في الشكل 1. مجموع الوقت الإجرائي هو أقل من 30 دقيقة. وتم الحصول على عينات الأنسجة باستخدام 6 ملم خزعة لكمة. كان وزن النسيج 0.18 ± 0.0...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

يوصف طريقة لعزل السريع الميتوكوندريا الخزعات النسيجية الثدييات. تم المتجانس كبد الفئران أو مستحضرات العضلات والهيكل العظمي مع dissociator النسيج التجاري وتم عزل الميتوكوندريا عن طريق الترشيح من خلال مرشحات التفاضلي شبكة من النايلون. الميتوكوندريا الوقت العزلة هو &lt;30 دقيقة مقارنة ب 60-100 دقيقة باستخدام طرق بديلة.

عزل السريع وتنقية الميتوكوندريا لزرع الأنسجة بواسطة التفكك والتفضيلية الترشيح

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Research

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Biology

37.8K Views.  Beth Israel Deaconess Medical Center and Harvard Medical School.  يوصف طريقة لعزل السريع الميتوكوندريا الخزعات النسيجية الثدييات. تم المتجانس كبد الفئران أو مستحضرات العضلات والهيكل العظمي مع dissociator النسيج التجاري وتم عزل الميتوكوندريا عن طريق الترشيح من خلال مرشحات التفاضلي شبكة من النايلون. الميتوكو...

Video: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

العزلة وزرع الخلايا الجذعية المكونة للدم (HSCs)

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

تنقية الميتوكوندريا من خلايا الخميرة

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

عزل وتنقية ذبابة الفاكهة الخلايا العصبية الطرفية بواسطة حبة فرز المغناطيسي

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

لونين عزل من قبل تنقية الحمض النووي الريبي (السقسقة)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

تجميد مستورقة، التشعيع جزئي، والنسج

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

العزلة والثقافة، وزرع خلايا العضلات الأقمار الصناعية

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

عزل الترشيح من الأحماض النووية: وبسيطة وسريعة استخراج الحمض النووي الطريقة

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.