저자는 그녀의 기술 지원 및 수행 조직학에 대한 크리스티안 Pahrmann 감사합니다.

<html> 8~12주의 나이에 20g의 최소 무게 Balb / C 마우스는 찰스 리버 (Sandhofer 웨그 7 D - 97633 Sulzfeld)에서 구입할 수 있습니다. 마우스는 일반 조건, 공급 표준 마우스 알약와 물 광고 libitum 아래에 위치해 있습니다. 유도 챔버를 사용하여 (2 %) isoflurane와 마우스를 마취. <ol><li> 목 지역 ribcage의 왼쪽을 면도하고 80 % 에탄올을 사용하는 소독. </li><li> 의 뒷면에있는 마우스를 놓고 마취를 유지하기 위해 코와 입을 통해 안면을 넣으십시오. </li><li> 마우스가 충분 anesthesized되었는지 확인하는 꼬리와 뒷다리를 곤란하게 반사를 확인합니다. </li><li> 미세한보기에서 호흡 관을 덮고있는 피부, 근육과 조직을 분리 정중선 자궁 절개를 수행합니다. </li><li> 기관은 endotracheal 튜브 (그림 1)을 삽입하는 성문 밑에 두 카트리지 고리 사이의 조직에 작은 구멍을 뚫고 노출하는 것입니다. 마이크로 외과 forecepts를 사용하여 기관의 두개골 부분을 잡고 endotracheal 튜브를 삽입합니다. 양쪽 폐에서 통풍이 잘되었는지 확인하는 흉부 움직임을 확인합니다. </li><li> 호흡 속도 (RR)은 17~18센티미터 H 2 O.의 inspiratory 압력으로 약 110 분당해야합니다 그 왼쪽에 직면하고, 그 오른쪽에 누워, 신중하게 마우스를 켭니다. 3 차 및 4 번째 갈비뼈 사이 leftsided thoracotomy를 수행하고, 출혈을 방지하기 위해 cauter을 사용하여 조심스럽게 조직 및 근육을 절개하다. </li><li> 조심스럽게 흉부를 열고 흉부가 열릴되면, 어떤 날카로운 물체로 폐을 건드리지 않고, 마음을 찾으십시오. 이제 마음을 덮고있는 pericardial 색의 일부를 제거합니다. </li><li> 그 소년은 폐동맥 및 좌측 외이 사이에 위치하고 있습니다. 하나의 봉합사 (그림 2)와 근위 젊은이 ligate에 8-0 Prolene 봉합사를 (Ethicon, Norderstedt, 독일)를 사용합니다. </li><li> 4 일 및 5 일 리브 사이의 가슴 튜브 (28G, 돈으로 좌우되는 카테터), 놓습니다. </li><li> 피부를 닫습니다 갈비뼈 4-0 Prolene 실행 봉합 (Ethicon, Norderstedt, 독일) 적응 6-0 Prolene 실행 봉합 (Ethicon, Norderstedt, 독일)를 사용하여, 레이어에서 흉부 절개를 닫습니다. </li><li> 신중하게 2ml 주사기 (그림 3)의 도움으로 흉부 드레인. 의 뒷면에있는 마우스를 놓습니다. 이제 endotracheal 튜브를 꺼내 7-0 Prolene의 봉합을 (Ethicon, Norderstedt, 독일)를 사용하여 하나의 스티치와 함께 tracheal 카트리지 반지를 적응. </li><li> 마우스의 얼굴 마스크를 놓고 봉합 (Ethicon, Norderstedt, 독일)을 실행하고 4-0 Prolene을 사용하여 피부를 닫습니다. </li></ol> 확인 내사 - 결합의 성공을 확인하는 다른 가능성이 있습니다. 트로포닌 검사는 동물을 안락사시키려는 필요없이 150 밖에 내 혈액을 사용하여 수술 후 6 때까지 18 시간 수행할 수 있습니다. 혈액은 맞춤 트로포닌 검사 키트 (파악 T 감성적, 로체, 만하임) (그림 4)에 적용됩니다. 트로포닌은 근육 세포의 굴지의 필라멘트에서 규정하는 단백질이며, 근육의 세 가지 유형의 다른 isotypes있다. isotypes cTnI와 cTnT는 심장에서 발견되고 조직 부상에 출시하고 있습니다. 트로포닌 T (cTnT)에 대한 표준 시험은 두 마음이 특정 단클론 항체 하나의 함정 혈액 샘플에서 트로포닌 기반으로, 두 번째는 마커입니다. infarcted 지역은 삼일 (그림 5) 이후 macroscopically 확인할 수 있습니다. TTC (2,3,5 - Triphenyltetrazolium 염화) 얼룩 대책 조직 생존이 경색 크기를 평가하는 데 사용됩니다. 에번스 청색 색소 (1.5 %, 1.0mL)에 인산 버퍼 호수 (PBS)가 심근 허혈성 영역을 측정하는 왼쪽 심실 캐비티에 주입합니다. 마우스는 euthanized되고 마음이 조각에 수확하고 sectioned입니다. 조직의 조각은 20 분에 대해 37 ° C에서 1퍼센트 TTC PBS 용액, pH를 7.4에서 incubated입니다. 조직은 2-8에서 하룻밤 PBS 버퍼 포르말린 10 % 해결된 ° C. TTC는 염색 에반스 블루 부정적인 영역 (그림 6) 전 (前) 생체내을 관리합니다. 조직학의 경우 마우스 euthanized 수 있으며 마음이 더 처리를위한 내장되어야한다. 파라핀 섹션을 수행 후, 슬라이드는 H &amp; E (hematoxylin 및 eosin) 물들일 또는 fibrotic 조직 (그림 7)를 시각화하는 trichrome 있습니다. <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="그림 1" /> 그림 1 : Tracheal 튜브. 1A : 노출 기관, 복부 자궁 절개 후. 1B : 기관은 후두에 꼬리 두 연골의 고리를 열립니다. 1C : Tracheal 튜브가 기관에 삽입됩니다. <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="그림 2" /> 그림 2 : 심장은 왼쪽 단면 thoracotomy를 통해 노출, 그 소년이 출혈도 잡았입니다. t &quot;&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="그림 3" /> 그림 3 : 흉부가 폐쇄되면 늑막 드레인이 수행됩니다. 마우스가 마취하에 아직도있다. <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="그림 4" /> 그림 4 : 트로포닌 검사가 긍정적인 경우 두 라인이 몇 분 후에 나타납니다. <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="그림 5" /> 그림 5 : explanted 마음을 표시합니다. 5A : 어두운 약간 흰 색상과 허혈성 면적 마음의 매크로 볼 수 있습니다. 5B : infarcted 마음의 단면, 허혈성 지역은 오른쪽 상단 모서리에 표시됩니다.

<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>

기금 현재의 연구는 대학 병원 함부르크 (MK, 여자 Habilitation 그랜트), DFG (SS, SCHR992/3-1), ISHLT (TD, 연구 대상 2009 년)에 의해 지원되었다. 성명서 모든 동물은 실험실 동물 관리의 원칙을 준수 인간의 치료를받은, 동물 실험에 의해 출판 연구소 동물의 관리 및 사용을위한 의료 연구 및 설명서에 대한 국립 협회가 정한 지침과 규정에 따라 수행되었습니다 보건 국립 연구소 (보건 간행물 85-23 국립 연구소, 1985 개정). 모든 동물은 찰스 리버 연구소 (Sulzfeld, 독일)에서 얻은되었고 대학 병원 함부르크 Eppendorf의 동물 보호 시설에서 유지되었다. 동물 표준 차우와 물 광고 libitum을 받았습니다.

요약이 비디오는 소년 - 결합 모델을 단계별로 단계를 시각화하고 쉽게 가능 수술 절차로 생쥐에서 내사 - ligations를 식별합니다.

lad-ligation: a murine model of myocardial infarction

경색 및 심근 국소 빈혈의 연구 모델은 심근 국소 빈혈의 급성 및 만성 pathobiological 및 pathophysiological 프로세스를 조사하고 향후 치료를 개발하고 최적화하는 데 필수적입니다. 심근 국소 빈혈을 만드는 두 가지 방법은 실험실 설치류에서 수행됩니다. 첫 번째 방법은 cryo - 펜은 심장 (1-3)의 표면에 적용 cryo 경색, 빠른 있지만 부정 확한 기술을 만드는 것입니다. 이 방법을 사용 과학자는 cryo - 흉터 또한 광대한 심근 손상은 심근 경색과 관련되지 않은 pathophysiological 부작용을 보여줍 만들어집니다, 국소 빈혈로 연결되는 보장할 수 없습니다. 두 번째 방법은 왼쪽 앞쪽에 내림차순 동맥 (내사)의 영구적인 결합입니다. 여기 내사 거의 즉시 볼 수 있습니다 국소 빈혈을 형성 하나의 스티치와 함께 출혈도 잡았입니다. 주변의 심근 조직이 거의 영향을받지 않는 상태에서 젊은이 폐쇄하여 더 이상 혈액의 흐름이 그 지역에서 허용되지 않습니다. 이 수술은 경색 심근 국소 빈혈 관련에서 발생 pathobiological 및 pathophysiological 측면을 imitates. 이 동영상에서 소개하는 방법은 젊은이 ligating하여 마우스 경색 모델의 수술을 보여줍니다. 이 모델은 심장 경색에 pathobiological 및 pathophysiological뿐만 아니라 immunobiological 연구 편리합니다. 표시 기술은 높은 정확도를 제공하며, histological 부분과 잘 연결합니다.

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

이 동영상은 심근 국소 빈혈의 pathobiological 및 pathophysiological 프로세스를 연구하기위한 빠르고 안정적​​인 모델을 사용하는 방법을 보여줍니다.

내사 - 결합 : 심근 경색의 Murine 모델

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

lad-ligation-a-murine-model-of-myocardial-infarction

Nanyang Technological University

Research

JoVE Journal

Biology

44.4K 조회수.  University Heart Center Hamburg. 이 동영상은 심근 국소 빈혈의 pathobiological 및 pathophysiological 프로세스를 연구하기위한 빠르고 안정적​​인 모델을 사용하는 방법을 보여줍니다.

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

Murine Pancreatic Islet Isolation

Murine 췌장 아일릿 격리

연구

생물학

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.

Hindlimb 국소 빈혈의 Murine 모델

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.

Murine 신장 이식 절차

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.

의 효율적인 생산 및 정제 재조합 쥐과 Kindlin-3 생물 물리학 연구 곤충 세포에서

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.

쥐 관상 동맥 혈관 평활근 세포의 분리

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.

심근 성능 및 산소 소비의 연구에 대한 설치류 작업 심장 모델

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.

Murine 콜린 불충분 한, Ethionine 보충 (CDE) 다이어트 만성 간 상해의 모델

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.

수직 소매 위절제술의 Murine 모델

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.

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.

진동절단 심근슬라이스에서 인간 심실 심근세포의 분리

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