Die Autoren danken Christiane Pahrmann für ihre technische Unterstützung und Durchführung der Histologie.

<html> Balb / C Mäuse mit einem Gewicht von mindestens 20g im Alter von 8 bis 12 Wochen sind von Charles River (Sandhofer Weg 7, D-97633 Sulzfeld) erworben. Mäuse sind unter üblichen Bedingungen, gefüttert Standard-Maus-Pellets und Wasser ad libitum untergebracht. Anesthetize Maus mit Isofluran (2%) mit einer Induktion Kammer. <ol><li> Shave Halsbereich und die linke Seite des Brustkorbs und desinfizieren mit 80% Ethanol. </li><li> Platzieren Sie die Maus auf den Rücken und legen Sie eine Gesichtsmaske über seinen Mund und Nase zu halten die Anästhesie. </li><li> Überprüfen Sie die Reflexe Einklemmen des Schwanzes und Hinterpfoten, um sicherzustellen, dass die Maus ausreichend narkotisiert ist. </li><li> Unter mikroskopischer Sicht führen Sie eine Mittellinie zervikale Inzision Trennung der Haut, Muskeln und Gewebe für die Luftröhre. </li><li> Wenn die Luftröhre freigelegt schneiden ein kleines Loch in das Gewebe zwischen zwei Patronen Ringe unterhalb der Glottis bis zu den Endotrachealtubus (Abbildung 1) einzufügen. Legen Sie die Endotrachealtubus Halten der kraniale Teil der Luftröhre mit mikrochirurgischen forecepts. Überprüfen Sie die Brust Bewegung sicher sein, dass beide Lungenflügel gut belüftet sind. </li><li> Die Atemfrequenz (RR) sollte etwa 110 pro Minute, mit einer inspiratorischen Druck von 17 bis 18cm H 2 O. Drehen Sie die Maus vorsichtig, liegt auf der rechten Seite, mit Blick auf die linke Seite. Führen Sie eine leftsided Thorakotomie zwischen dem 3. und der 4. Rippe und sezieren das Gewebe und Muskeln vorsichtig mit einem Kauter, um Blutungen zu verhindern. </li><li> Öffnen Sie den Brustkorb sorgfältig, nachdem der Brustkorb geöffnet wird, finden die Herzen, ohne die Lunge mit einem spitzen Gegenstand. Entfernen Sie nun den Teil der Herzbeutel, die für das Herz ist. </li><li> Die LAD ist zwischen Lungenarterie und dem linken Vorhof befindet. Verwenden Sie einen 8-0 Prolene Naht (Ethicon, Norderstedt, Deutschland), um die LAD proximal mit einer einzigen Naht (Abb. 2) zu unterbinden. </li><li> Legen Sie eine Thoraxdrainage (28G, käuflich Katheter), zwischen dem 4. und dem 5. Rippe. </li><li> Schließen Sie die Brust Einschnitt in Schichten mit 6-0 Prolene fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland) an den Rippen und 4:0 Prolene fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland) Adaption auf der Haut zu schließen. </li><li> Lassen Sie den Brustkorb mit Hilfe einer Spritze 2ml sorgfältig (Abbildung 3). Platzieren Sie die Maus auf dem Rücken. Jetzt nimmt man den Tubus aus und passen Sie die tracheale Patrone Ringe mit einem einzigen Stich mit 7-0 Prolene Fäden (Ethicon, Norderstedt, Deutschland). </li><li> Legen Sie die Maske auf der Maus und schließen Sie die Haut mit 4-0 Prolene fortlaufenden Nähten (Ethicon, Norderstedt, Deutschland). </li></ol> Bestätigung Es gibt verschiedene Möglichkeiten, um den Erfolg des LAD-Ligation zu bestätigen. Der Troponin-Test kann von 6 bis 18 Stunden nach der Operation durchgeführt werden, mit nur 150 l Blut, ohne die Notwendigkeit, das Tier einzuschläfern. Das Blut wird auf eine individuelle Troponin-Test-Kit (TROP T Sensitive, Roche, Mannheim) (Abbildung 4). Troponin ist ein regulative Protein in der Aktin-Filamente der Muskelzelle, gibt es verschiedene Isotypen in den drei Arten von Muskeln. Die Isotypen cTnI und cTnT sind im Herzen gefunden und sind in Gewebeverletzung freigesetzt. Der standardisierte Test für Troponin T (cTnT) auf zwei Herzen spezifischen monoklonalen Antikörper, einer fängt die Troponin in der Blutprobe basiert, ist der zweite Marker. Das Infarktareal kann makroskopisch nach 3 Tagen (Abbildung 5) identifiziert werden. TTC (2,3,5-Triphenyltetrazoliumchlorid) Färbung Maßnahmen Gewebeviabilität zur Infarktgröße zu bewerten. Evans blauen Farbstoff (1,5%, 1,0 ml) in Phosphat-gepufferter Salzlösung (PBS) in den linken Ventrikel injiziert, um den myokardialen ischämischen Bereich zu messen. Die Maus ist eingeschläfert und das Herz wird geerntet und geschnitten in Scheiben schneiden. Die Gewebeschnitte werden in 1% TTC PBS-Lösung, pH 7,4 bei 37 ° C für 20min inkubiert. Die Gewebe werden in 10% PBS-gepuffertem Formalin über Nacht bei 2-8 ° C fixiert TTC wird ex vivo zu färben Evans blue-negative Bereiche (Abb. 6) verabreicht werden. Für die Histologie der Maus muss eingeschläfert werden und das Herz hat für die weitere Verarbeitung eingebettet werden. Nach der Durchführung Paraffinschnitte werden Objektträger mit H &amp; E (Hämatoxylin und Eosin) gefärbten oder Trichrom zu Bindegewebe (Abb. 7) zu visualisieren. <img src="/files/ftp_upload/1438/1438fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Trachealtubus. 1A: Exposed Luftröhre, nach ventral zervikalen Inzision. 1B: Trachea ist zwei Knorpelspangen kaudal des Kehlkopfes eröffnet. 1C: Trachealtubus in die Luftröhre eingeführt. <img src="/files/ftp_upload/1438/1438fig2.jpg" alt="Abbildung 2" /> Abbildung 2: Das Herz durch eine linksseitige Thorakotomie ausgesetzt ist, die LAD ligiert. t &quot;&gt; <img src="/files/ftp_upload/1438/1438fig3.jpg" alt="Abbildung 3" /> Abbildung 3: Nach dem Brustkorb geschlossen wird, eine Pleura drain durchgeführt wird. Die Maus ist immer noch unter Narkose. <img src="/files/ftp_upload/1438/1438fig4.jpg" alt="Abbildung 4" /> Abbildung 4: Zwei Linien erscheinen nach ein paar Minuten, wenn die Troponin-Test ist positiv. <img src="/files/ftp_upload/1438/1438fig5.jpg" alt="Abbildung 5" /> Abbildung 5: Zeigt die explantierten Herzen. 5A: Makroskopische Ansicht des Herzens mit den ischämischen Bereich mit einem verschossenen weißliche Farbe. 5B: Querschnitt durch den Infarkt Herz, zeigt den ischämischen Bereich in der oberen rechten Ecke.

<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>

Finanzierung Die vorliegende Studie wurde von der Universitätsklinik Hamburg (MK, Frau Habilitation Grant), DFG (SS, SCHR992/3-1), ISHLT (TD, Research Award 2009) unterstützt. Aussage Alle Tiere erhielten menschlicher Obhut in Übereinstimmung mit den Prinzipien des Laboratory Animal Care, wurden Experimente an Tieren in Übereinstimmung mit den Richtlinien und Vorschriften her von der Nationalen Gesellschaft für Medizinische Forschung und der Leitfaden für die Pflege und Verwendung von Labortieren durch die veröffentlichten durchgeführt National Institutes of Health (National Institutes of Health Veröffentlichung 85-23, revised 1985). Alle Tiere wurden von Charles River Laboratories (Sulzfeld, Deutschland) erhalten und wurden in der Tierpflege Einrichtungen des Universitätsklinikums Hamburg Eppendorf gepflegt. Die Tiere erhielten Standard-Futter und Wasser ad libitum.

Zusammenfassend visualisiert dieses Video der LAD-Ligatur-Modell Schritt-für-Schritt identifiziert und LAD-Ligation in Mäusen als leicht durchführbar chirurgischen Eingriffen.

lad-ligation: a murine model of myocardial infarction

Forschung Modelle der Infarkt und Ischämie sind unerlässlich, um die akute und chronische pathobiologischen und pathophysiologischen Prozessen in Myokardischämie zu untersuchen und zu entwickeln und zu optimieren künftige Behandlung. Zwei verschiedene Methoden zur Erstellung Myokardischämie sind in Labornagern durchgeführt. Die erste Methode ist die Kryo-Infarkt, eine schnelle, aber ungenaue Methode, wo ein Kryo-Stift auf der Oberfläche des Herzens (1-3) angewendet wird, zu schaffen. Mit dieser Methode kann der Wissenschaftler nicht garantieren, dass die Kryo-Narbe Ischämie führt, auch eine große Myokardschäden erstellt, zeigt pathophysiologischen Nebenwirkungen, die nicht auf Herzinfarkt in Zusammenhang stehen. Die zweite Methode ist die permanente Ligation der linken vorderen absteigenden Arterie (LAD). Hier werden die LAD ist mit einem einzigen Stich ligiert und bilden eine Ischämie, die fast sofort gesehen werden kann. Durch das Schließen der LAD, sind keine weiteren Blutfluss in diesem Bereich nicht erlaubt, während die umgebenden Herzmuskelgewebe ist fast nicht betroffen. Dieses chirurgische Verfahren ahmt die pathobiologischen und pathophysiologische Aspekte auftretenden Infarkt-bezogenen Myokardischämie. Die Methode in diesem Video vorgestellt zeigt den chirurgischen Eingriff einer Maus Infarkt Modell durch Ligation der LAD. Dieses Modell ist für pathobiologischen und pathophysiologischen sowie immunbiologische Studien über Herzinfarkt bequem. Die gezeigten Technik bietet eine hohe Genauigkeit und korreliert gut mit histologischen Schnitten.

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

Dieses Video zeigt, wie eine schnelle und zuverlässige Modell nutzen, um pathobiologischen und pathophysiologischen Prozessen der Myokardischämie zu studieren.

LAD-Ligation: einem Mausmodell der Myokardinfarkt

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 Aufrufe.  University Heart Center Hamburg. Dieses Video zeigt, wie eine schnelle und zuverlässige Modell nutzen, um pathobiologischen und pathophysiologischen Prozessen der Myokardischämie zu studieren.

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

Murine Pancreatic Islet Isolation

Murine Inselzellen der Bauchspeicheldrüse Isolation

Forschung

Biologie

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.

Mausmodell der hinteren Gliedmaßen Ischämie

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 Renal Transplantation

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.

Effizienten Herstellung und Reinigung von rekombinantem murinem Kindlin-3 von Insektenzellen für biophysikalische Studien

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.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

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.

Nagetier Arbeits Herz Modell für die Untersuchung der myokardialen Leistung und Sauerstoffverbrauch

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.

Der murinen Cholin-Mangel, Ethionine ergänzt (CDE) Diät-Modell einer chronischen Leberschädigung

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.

Ein Mausmodell der vertikalen Sleeve-Gastrektomie

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.

Massenspektrometrieanalyse zur Identifizierung der Ubiquitylierung von EIFP-A mit EYFP-Tags (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.

Isolierung menschlicher ventrikulärer Kardiomyozyten aus Vibratome-Cut Myokardscheiben

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