الكتاب أشكر كريستيان Pahrmann لمساعدتها التقنية والأنسجة الأداء.

<html> يتم شراؤها BALB / C الفئران وزنها يقل عن 20G في سن 8 إلى 12 أسبوعا من نهر تشارلز (7 Sandhofer خبراء المياه ، D - 97633 Sulzfeld). ويعيش في ظل ظروف الفئران التقليدية ، والكريات تغذية الماوس القياسية والمياه بالمال وبالشهرة أيضا الإعلانية. تخدير الفأر مع isoflurane (2 ٪) باستخدام غرفة التعريفي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حلق منطقة الرقبة والجانب الأيسر من القفص الصدري وتعقيمها باستخدام الايثانول 80 ٪. </li><li style=";text-align:right;direction:rtl"> ضع الماوس على ظهرها ووضع قناع على الأنف والفم والخمسين للحفاظ على التخدير. </li><li style=";text-align:right;direction:rtl"> التحقق من ردود الفعل معسر الذيل والقدمين الخلفيتين للتأكد من أن الفأر هو anesthesized كافية. </li><li style=";text-align:right;direction:rtl"> تحت عرض المجهرية إجراء شق خط الوسط عنق الرحم التي تفصل بين الجلد والعضلات والأنسجة التي تغطي القصبة الهوائية. </li><li style=";text-align:right;direction:rtl"> عندما يتعرض القصبة الهوائية قطع ثقب صغير في الأنسجة بين عصابات خرطوشة جهازي أدناه على المزمار لادخال الأنبوب الرغامي (الشكل 1). إدراج أنبوب القصبة الهوائية عقد جزء من الجمجمة القصبة الهوائية باستخدام forecepts الجراحية الصغرى. تحقق من حركة الصدر للتأكد من أن تكون جيدة التهوية كلا الرئتين. </li><li style=";text-align:right;direction:rtl"> يجب أن معدل التنفس (RR) أن ما يقرب من 110 في الدقيقة ، مع الضغط الشهيقي من 17 إلى 18cm H 2 O. بدوره الماوس بعناية ، والكذب على جانبها الأيسر ، تواجه جانبها الأيسر. أداء بضع الصدر leftsided بين الثالثة (3) والضلع الرابع 4 ، وتشريح الانسجة والعضلات بعناية ، وذلك باستخدام مكواة لمنع النزيف. </li><li style=";text-align:right;direction:rtl"> فتح القفص الصدري بعناية ، وبمجرد فتح القفص الصدري ، والعثور على القلب والرئة من دون لمس أي جسم مع حادة. الآن إزالة جزء من كيس التامور الذي يغطي القلب. </li><li style=";text-align:right;direction:rtl"> يقع LAD بين الشريان الرئوي والأذن اليسرى. استخدام 8-0 Prolene خياطة (Ethicon ، Norderstedt ، المانيا) لligate الفتى القريبة مع واحد وحيد خياطة (الشكل 2). </li><li style=";text-align:right;direction:rtl"> وضع أنبوب الصدر (28G ، القسطرة المرتشين) ، في الفترة الواقعة بين 4 و 5 الضلع الرابع. </li><li style=";text-align:right;direction:rtl"> إغلاق شق الصدر في الطبقات ، وذلك باستخدام الخيوط 6-0 Prolene تشغيل (Ethicon ، Norderstedt ، المانيا) للتكيف مع الأضلاع والغرز Prolene 4-0 تشغيل (Ethicon ، Norderstedt ، المانيا) لإغلاق الجلد. </li><li style=";text-align:right;direction:rtl"> استنزاف الصدر بمساعدة حقنة 2ml بعناية (الشكل 3). ضع الماوس على ظهرها. تتخذ الآن من الأنبوب الرغامي وتكييف حلقات القصبة الهوائية خرطوشة واحدة واحدة باستخدام غرزة خياطة الجروح Prolene 7-0 (Ethicon ، Norderstedt ، ألمانيا). </li><li style=";text-align:right;direction:rtl"> وضع قناع للوجه على الماوس وإغلاق الجلد باستخدام Prolene 4-0 تشغيل الغرز (Ethicon ، Norderstedt ، ألمانيا). </li></ol> التحقق من صحة هناك احتمالات مختلفة لتأكيد نجاح عملية ربط - LAD. ويمكن إجراء الاختبار تروبونين 6-18 ساعة بعد الجراحة ، وذلك باستخدام سوى 150 لتر الدم من دون الحاجة إلى الموت ببطء الحيوان. يتم تطبيق الدم لعدة اختبار تروبونين مخصصة (T TROP الحساسة ، روش ، مانهايم) (الشكل 4). تروبونين هو بروتين التنظيمية في شعيرات أكتين من الخلية العضلات ، وهناك isotypes مختلفة في ثلاثة أنواع من العضلات. تم العثور على cTnI isotypes وcTnT في قلب وصدر في إصابة الأنسجة. ويستند اختبار موحد لT تروبونين (cTnT) على اثنين من الأجسام المضادة وحيدة النسيلة القلب محددة ، واحدة من الفخاخ تروبونين في عينة من الدم ، والثانية هي علامة. ويمكن تحديد المنطقة احتشاء ظاهريا بعد 3 أيام (الشكل 5). TTC (2،3،5 - Triphenyltetrazolium كلوريد) الأنسجة تدابير السلامة تلطيخ المستخدمة لتقييم حجم احتشاء. صبغة زرقاء ايفانز (1،5 ٪ ، 1.0mL) في الفوسفات مخزنة يتم حقن المياه المالحة (PBS) في تجويف البطين الأيسر لقياس مساحة العضلة القلبية الدماغية. الموت الرحيم هو الماوس ويتم حصاد القلب ومقطوع إلى شرائح. يتم تحضين شرائح الأنسجة في 1 ٪ حل PBS TTC ، ودرجة الحموضة 7.4 عند 37 درجة مئوية لمدة 20min. يتم إصلاح الأنسجة في برنامج تلفزيوني ، 10 ٪ بين عشية وضحاها مخزنة في الفورمالين 2-8 درجة مئوية. يدار TTC فيفو السابقين إلى المناطق صبغة زرقاء السلبية ايفانز (الشكل 6). لالأنسجة الماوس يجب أن يكون الموت الرحيم والقلب يجب أن تكون جزءا لا يتجزأ من لمزيد من المعالجة. بعد أداء أقسام البارافين ، ملطخة الشرائح مع H &amp; E (الهيماتوكسيلين ويوزين) أو ثلاثي الألوان لتصور نسيج متليف (الشكل 7). <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="الشكل 1" /> الشكل 1 : أنبوب القصبة الهوائية. 1A : مكشوف القصبة الهوائية ، وبعد شق بطني عنق الرحم. 1B : فتح القصبة الهوائية حلقات الغضروف two الذيلية إلى الحنجرة. 1C : يتم إدخال أنبوب القصبة الهوائية في القصبة الهوائية. <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="الشكل 2" /> الشكل 2 : يتم كشفها من خلال بضع الصدر والقلب a الجانب الأيسر ، وligated الفتى. ر &quot;&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="الشكل 3" /> الشكل 3 : بعد إغلاق الصدر ، يتم تنفيذ عملية استنزاف الجنبة. الماوس لا يزال تحت التخدير. <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="الشكل 4" /> الشكل 4 : خطان تظهر بعد بضع دقائق عند الاختبار تروبونين إيجابية. <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="الشكل 5" /> الشكل 5 : يعرض explanted القلب. 5A : عرض العينية من القلب مع المنطقة الدماغية مع اللون بيضاء باهتة. 5B : الباب الصليب احتشاء القلب ، وهي المنطقة الدماغية يظهر في الزاوية اليمنى العليا.

<ol>
	<li>Atkins, B. Z., Hueman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+diastolic+properties+of+injured+Heart.">Cellular cardiomyoplasty improves diastolic properties of injured Heart.</a> J Surg Res. 85 (2), 234-242 (1999).</li><li>Jaquet, K., KT, K. r. a. u. s. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+myocardial+scar+size+after+implantation+of+mesenchymal+stem+cells+in+rats:+what+is+the+mechanism.">Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism.</a> Stem Cells Dev. 14 (3), 299-309 (2005).</li><li>Joung, B., Kim, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+mononuclear+stem+cells+transplanted+in+rat+infarct+myocardium+improved+the+electrical+conduction+without+evidence+of+proarrhythmic+effects.">Bone marrow mononuclear stem cells transplanted in rat infarct myocardium improved the electrical conduction without evidence of proarrhythmic effects.</a> Yonsei Med J. 48 (5), 754-764 (2007).</li></ol>

التمويل وأيد هذه الدراسة من جامعة هامبورغ مستشفى (MK ، المرأة التأهيل غرانت) ، DFG (SS ، SCHR992/3-1) ، ISHLT (TD ، وجائزة البحوث 2009). بيان وكانت جميع الحيوانات تلقوا رعاية الإنسان وفقا للمبادئ رعاية الحيوان المعملية ، وأجرى تجارب على الحيوانات وفقا للمبادئ التوجيهية واللوائح التي وضعتها الجمعية الوطنية للبحوث الطبية ودليل لرعاية واستخدام الحيوانات المختبرية التي نشرتها المعاهد الوطنية للصحة (المعاهد الوطنية للصحة المنشور 85-23 ، منقحة 1985). تم الحصول على جميع الحيوانات من مختبرات نهر تشارلز (Sulzfeld ، ألمانيا) ، وكان يحتفظ بها في مرافق الرعاية الحيوانية من مستشفى جامعة هامبورغ إيبندورف. تلقى طعام الحيوانات والمياه القياسية بالمال وبالشهرة أيضا الإعلانية.

باختصار ، هذا الفيديو يتصور النموذج LAD - ربط خطوة بخطوة ، ويحدد - LAD عملية ربط في الفئران والعمليات الجراحية ممكنا بسهولة.

lad-ligation: a murine model of myocardial infarction

النماذج البحثية لاحتشاء عضلة القلب ونقص التروية ضرورية للتحقيق في العمليات الحادة والمزمنة وpathobiological الفيزيولوجية المرضية في نقص التروية القلبية وتطوير وتحسين العلاج في المستقبل. يتم تنفيذ اثنين من أساليب مختلفة من نقص تروية عضلة القلب في إنشاء مختبر القوارض. الأسلوب الأول هو خلق احتشاء البرد ، وهي تقنية سريعة ولكن غير دقيقة ، حيث يتم تطبيق البرد القلم على سطح القلب (1-3). باستخدام هذه الطريقة يمكن للعالم لا تضمن أن البرد يؤدي إلى نقص التروية ندبة ، كما يتم إنشاء إصابة عضلة القلب الشاسعة التي تظهر آثار جانبية الفيزيولوجية المرضية التي لا علاقة لها احتشاء عضلة القلب. الأسلوب الثاني هو ربط دائم من الشريان الأيسر الأمامي النازل (LAD). هنا ligated الفتى غرزة واحدة واحدة ، وتشكيل نقص التروية التي يمكن رؤيتها على الفور تقريبا. عن طريق إغلاق LAD ، لا يسمح مزيد من تدفق الدم في هذا المجال ، في حين أن ما يقرب من الأنسجة المحيطة لا تتأثر عضلة القلب. هذا الإجراء الجراحي يقلد الجوانب pathobiological والفيزيولوجية المرضية التي تحدث في احتشاء عضلة القلب المرتبطة نقص التروية. طريقة عرض في هذا الفيديو يوضح الإجراء الجراحي للماوس نموذج احتشاء ligating بواسطة الفتى. هذا النموذج هو مناسب للدراسات pathobiological والفيزيولوجية المرضية وكذلك المناعية على احتشاء عضلة القلب. أظهرت هذه التقنية توفر الدقة العالية ويرتبط بشكل جيد مع المقاطع النسيجية.

Watch this Scientific Journal Video about LAD-Ligation: A Murine Model of Myocardial Infarction at JoVE.com

LAD-Ligation: A Murine Model of Myocardial Infarction

هذا الفيديو يوضح كيفية استخدام نموذج سريعة وموثوق بها لدراسة العمليات الفيزيولوجية المرضية وpathobiological من نقص تروية عضلة القلب.

LAD - الربط : نموذج الفئران من احتشاء عضلة القلب

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

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Biology

44.4K Views.  University Heart Center Hamburg. هذا الفيديو يوضح كيفية استخدام نموذج سريعة وموثوق بها لدراسة العمليات الفيزيولوجية المرضية وpathobiological من نقص تروية عضلة القلب.

Video: LAD-Ligation: A Murine Model of Myocardial Infarction

Murine Pancreatic Islet Isolation

فأري عزل جزيرة البنكرياس

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

فأري موديل الإسكيمية Hindlimb

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

فأري إجراء زراعة الكلى

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

كفاءة الإنتاج وتنقية المؤتلف الفئران Kindlin-3 من خلايا الحشرات للدراسات البيوفيزيائية

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

عزل الفئران التاجي الأوعية الدموية خلايا العضلات الملساء

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

القوارض العمل نموذج القلب لدراسة أداء عضلة القلب واستهلاك الأوكسجين

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

مورين الكولين تعاني من نقص، تستكمل اثيونيني (الرفيق) النظام الغذائي النموذجي لإصابة الكبد المزمنة

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

نموذج موريني Gastrectomy الأكمام العمودي

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

تحليل قياس الطيف الكتلي لتحديد Ubiquitylation من EYFP الموسومة CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

عزل القلب البطيني البشري من شرائح عضلة القلب المقطّع بالهزاز

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...