Name: live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy
Uploaded: 2014-06-24T14:00:00.000+00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

We would like to thank Dr. Alengo Nyamay'antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.

<ol>
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The authors have nothing to disclose.

Primäre Herzmuskelzellen aus neugeborenen Ratten isoliert werden seit langem verwendet, um Funktionen Kardiomyozyten in vitro zu studieren. Dieses Protokoll beschreibt ein Verfahren zur Isolierung von neonatalen Herzmuskelzellen aus Rattenjungen mit einer zweistufigen Enzymverdauung Verfahren wird zunächst Verdau mit Trypsin O / N bei 4 ° C und anschließend gereinigten Kollagenase. Ein Vorteil der Verwendung des gereinigten Kollagenase Schritt ist, dass der gleiche Grad von Enzym wird für alle Isolierungen...

Primäre Ratten-Herzmuskelzellen Neugeborenen haben lange für die Untersuchung von Herzmuskelzellen in vitro Funktion 1 verwendet. Sie sind leicht zu von Rattenjungen von mehreren etablierten Methoden 4.2 zu isolieren. Das häufigste Verfahren verwendet Kollagenase oder Trypsin Bindegewebe des Herzens vor der Zellisolierung zu verdauen. Die Forscher haben auch Methoden zur Isolierung von Herzmuskelzellen aus adulten Nagetieren 5-8 sowie 9,10 neugeborenen Mäusen entwickelt. Dieses Protokoll beschreibt ein Verfahren zum Isolieren von Kardiomyozyten aus neonatalen Ratten-Jungtieren unter Verwendung einer zweistufigen Enzymverdauung Verfahren. Trypsin wird zuerst verwendet, O / N bei 4 ° C und anschließend gereinigten Collagenase bei 37 ° C. Inkubation des Herzgewebes mit Trypsin O / N bei 4 ° C reduziert die notwendigen Schritte Ernten der Zellen im Vergleich zu Verfahren mit sequentieller Inkubation in einem warmen Enzymlösung 2. Zusätzlich kann durch Verwendung von gereinigtem Collagenase anstatt rohe Enzyme können von Los zu Los-Variabilität eliminiert werden, wodurch eine verbesserte Reproduzierbarkeit. Funktionsstudien eines bestimmten Proteins verwenden oft ein Protein-Expressionssystem unter Verwendung eines Adenovirus, 11-13 und / oder ein Lentivirus 14-16. [WARN HINWEIS] Die Virusproduktion und Manipulation sollte nach den NIH-Richtlinien durchgeführt werden. Das Adenovirus nicht in das Wirtsgenom zu integrieren. Es hat einen sehr hohen Transduktionseffizienz in den meisten Zelltypen, einschließlich teilende Zellen und nicht-teilende Zellen, wie auch primäre Zellen und etablierten Zelllinien. Dies macht das Adenovirus eine zuverlässige Vektor zur Genexpression. Hohe Konzentrationen des Proteins von dem Adenovirus-Vektor codiert entwickeln innerhalb von 48 Stunden nach Transduktion, und sie für 17 Wochen dauern. Ein Nachteil der Verwendung eines Adenovirus für die Proteinexpression ist jedoch, daß die Entwicklung eines rekombinanten adenovirus ist sowohl kompliziert als auch zeitaufwendig. Dieser Nachteil erklärt zum Teil, warum viele Forscher Lentiviren worden Drehen für rekombinante Genexpression. Im Gegensatz zu adenoviralen Konstrukte, wodurch eine lentiviralen Konstrukt ist schnell und einfach. Obwohl Lentiviren haben geringere Effizienz der Transduktion als Adenoviren, in beiden Teilen und nicht-teilende Zellen, sie in das Wirtsgenom zu integrieren. Folglich ist die Expression des transduzierten Gens für Lentiviren als für Adenoviren stabiler. Aufgrund technologischer Fortschritte in der Mikroskopie, ist es viel einfacher, lebende Zellbilder von Zellen, die rekombinanten Proteine ​​zu erfassen. Dies gilt auch bei Videorate Geschwindigkeiten des Erwerbs. Dies ermöglicht dem Prüfer, um festzustellen, wie bestimmte Veränderungen in dem Protein von Interesse in funktioneller Einfluss auf die Zelle in Echtzeit. Das konfokale Spinning-Disk-Mikroskop hat mehrere Eigenschaften, die es eine optimale Technik für liv machenE-Cell-Imaging 18,19. Die Yokogawa Spinning Disc ermöglicht viel schnellere Bildaufnahme, während zur gleichen Zeit nutzen weit weniger Strom als Laserpunkt konfokalen Mikroskopen. Beide dieser Besonderheiten sind aufgrund der sich drehenden Scheibe, die eine Vielzahl Löcher, durch die konfokale Laser gelangt gleichzeitig an die Proben enthält. Während der Erwerb, die Festplatte selbst dreht sich schnell und kontinuierlich 18-20. Durch die Verwendung des Autofokus-Systems des Mikroskops wird eine stabile Fokus über lange Zeiträume aufrechterhalten. Diese ermöglicht es Forschern, gut fokussierte Bilder Live Cell nehmen. Erworbene Bilder werden als Filmdatei gespielt. Die Bilder werden mit Bildanalyse-Software wie ImageJ 21,22, FIJI 23 oder anderen im Handel erhältlichen Software analysiert.

<table><tbody><tr><td>1 Scissors for decapitation</td><td>WPI</td><td>501749</td><td>Autoclave before use</td></tr><tr><td>1 Fine scissors for heart isolation and chopping</td><td>WPI</td><td>14393</td><td>Autoclave before use</td></tr><tr><td>2 Fine forceps (Dumont No. 5)</td><td>Sigma</td><td>F6521</td><td>Autoclave before use</td></tr><tr><td>3 Sterilized 10 cm plastic dishes</td><td>Sigma</td><td>CLS430165</td><td>For heart isolation</td></tr><tr><td>3.5 cm Glass bottom culture dishes</td><td>MatTek</td><td>P35G-1.5-20-C</td><td>For final plating of cardiomyocytes for future live cell imaging. Micro-Dishes from ibidi are an acceptable alternative.</td></tr><tr><td>3.5 cm Glass bottom culture dishes</td><td>MatTek</td><td>P35G-0.17-14-C</td><td>For TIRF or high resolution image</td></tr><tr><td>Ethanol solution, 70% (v/v) in water</td><td>Sigma</td><td>E7148</td><td></td></tr><tr><td>2% Gelatin</td><td>Sigma</td><td>G1393</td><td></td></tr><tr><td>1 Sterilized 10 cm plastic dish</td><td>Sigma</td><td>CLS430165</td><td>For trypsinization</td></tr><tr><td>Aluminium foil</td><td>Any brand</td><td></td><td></td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7543</td><td></td></tr><tr><td>Two 10 cm plastic cell culture dish</td><td>Sigma</td><td>CLS430165</td><td>For selection</td></tr><tr><td>Autopipette</td><td>Drummond Scientific</td><td>4-000-300</td><td>For trituration</td></tr><tr><td>Cell counter</td><td></td><td></td><td></td></tr><tr><td>Cellometer, automated cell counter</td><td>nexcelom</td><td></td><td>To check and count cells</td></tr><tr><td>Microscope and hematocytometer</td><td>Any brand</td><td></td><td>To check and count cells</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>invitrogen</td><td>15250-061</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>MCO-19AIC</td><td></td></tr><tr><td>Incubating orbital shaker</td><td>Sigma</td><td>Z673129</td><td>To shake heart tissue with collagenase at 37 &amp;deg;C at 170-200 rpm</td></tr><tr><td>10 mg/ml BrdU solution</td><td>BD Pharmingen</td><td>550891</td><td></td></tr><tr><td>DMEM, high glucose</td><td>invitrogen</td><td>41965039</td><td>Mix medium as indicated in the protocol and warm before use</td></tr><tr><td>MEM</td><td>invitrogen</td><td>31095029</td><td>Mix medium as indicated in the protocol and warm before use</td></tr><tr><td>Fetal bovine serum</td><td>invitrogen</td><td>26140079</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/ml)&amp;nbsp;</td><td>invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Section 2 Lentiviral transduction</td><td></td><td></td><td></td></tr><tr><td>3 Packaging plasmids</td><td></td><td></td><td></td></tr><tr><td>pMDLg/pRRE</td><td>Addgene</td><td>12251</td><td></td></tr><tr><td>pRSV-Rev</td><td>Addgene</td><td>12253</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>The lentiviral transfer vector, pLVX-IRES-Puro</td><td>Clontech</td><td>632183</td><td></td></tr><tr><td>Opti-MEM (serum-free medium)</td><td>invitrogen</td><td>31985070</td><td></td></tr><tr><td>Transfection reagent</td><td></td><td></td><td></td></tr><tr><td>polyethyleneimine&amp;ldquo;Max&amp;rdquo;, (MW 40,000) - High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form)&amp;nbsp;</td><td>Polysciences</td><td>24765-2</td><td>It can be substituted with X-tremeGENE 9 from Roche</td></tr><tr><td>X-tremeGENE 9</td><td>Roche</td><td>6365779001</td><td>substitute for PEI as transfection reagent</td></tr><tr><td>Chloroquine</td><td>Sigma</td><td>C6628</td><td>Dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 &amp;mu;M Chloroquine.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>10 ml Luer-Lok syringe, sterilized</td><td>BD</td><td>309604</td><td></td></tr><tr><td>0.45 &amp;mu;m filters</td><td>Sigma</td><td>F8677</td><td>Use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus.</td></tr><tr><td>Hexadimethrine bromide</td><td>Sigma</td><td>H9268</td><td>Dissolve in water and make 8 mg/ml stock solution, then filter it to sterilize.</td></tr><tr><td>Polyethylene glycol 6,000</td><td>Sigma</td><td>81260</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Section 3 Adenoviral transduction</td><td></td><td></td><td></td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>Some 10 cm cell culture dishes</td><td>Sigma</td><td>CLS430165</td><td></td></tr><tr><td>96-Well microplate with lid, flat-bottom, tissue culture, sterile</td><td>BD Falcon</td><td>353072</td><td>For titration</td></tr><tr><td>Multichannel pipette, 10-100 &amp;mu;l, 8-channel</td><td>eppendorf</td><td>3122 000.035</td><td></td></tr><tr><td>Section 4 Live cell imaging</td><td></td><td></td><td></td></tr><tr><td>Spinning disk confocal microspopy</td><td>PerkinElmer</td><td>L7267000</td><td></td></tr><tr><td>A temperature-controlled chamber</td><td>Any brand</td><td></td><td>To keep temperature at 37 &amp;deg;C</td></tr><tr><td>A CO&lt;sub&gt;2&lt;/sub&gt; environmental system</td><td>Any brand</td><td></td><td>Optional to maintain CO&lt;sub&gt;2&lt;/sub&gt; concentration optimal</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Independent medium, no glutamine</td><td>invitrogen</td><td>18045-054&amp;nbsp;&amp;nbsp;</td><td>For long time time-lapse imaging</td></tr><tr><td>DMEM, high glucose, HEPES, no phenol red</td><td>invitrogen</td><td>21063-029</td><td>For long time time-lapse imaging</td></tr></tbody></table>

live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy

Primäre Ratten-Herzmuskelzellen Neugeborenen sind bei der Grundlagenforschung in vitro Herz-Kreislauf, weil sie leicht in großen Stückzahlen getrennt werden, in einem einzigen Verfahren. Aufgrund der Fortschritte in der Mikroskoptechnik ist es relativ einfach, Live-Cell-Bilder für die Zwecke der Untersuchung zellulärer Ereignisse in Echtzeit mit minimaler Besorgnis über Phototoxizität zu den Zellen zu erfassen. Dieses Protokoll beschreibt, wie man Live-Cell-Zeitrafferbilder von primären Ratten-Herzmuskelzellen nehmen Neugeborenen mit einem konfokalen Mikroskop Spinning-Disk folgende lentiviralen und adenovirale Transduktion, um die Eigenschaften der Zelle zu modulieren. Die Anwendung von zwei unterschiedlichen Arten von Viren macht es einfacher, eine geeignete Übertragungsrate und die Expressionsniveaus für zwei unterschiedliche Gene zu erreichen. Nun konzentriert Lebendzell Bilder können mit Autofokus-System des Mikroskops, die stabile Fokus für lange Zeiträume beibehält, erhalten werden. Gemäss dieser Methode werden die Funktionen von exogen Ingenieured Proteinen in kultivierten primären Zellen exprimiert werden, können analysiert werden. Zusätzlich kann dieses System verwendet, um die Funktionen von Genen durch die Verwendung von siRNAs sowie von chemischen Modulatoren zu untersuchen.

Die Technik, ein Lentivirus-Codierung EGFP (enhanced green fluorescent protein)-markierten Cx43 (Connexin43) oder eine mutierte Cx43 32 wurde verwendet, um EGFP-markierte Proteine ​​in Zellen zu exprimieren, und ein Adenovirus-Codierung FGFR1DN (Fibroblasten-Wachstumsfaktor-Rezeptor 1 dominant negative illustrieren ) wurde verwendet, um in der Zelle 33-35 heruntergefahren FGF-Signalisierung. Drei Tage nach der Trennung von Kardiomyozyten wurden die isolierten Kardiomyozyten mit Lentiviren, um E...

Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Live Cell Imaging von primären Ratten-Kardiomyozyten Neonatal Nach Adenovirale und Lentivirale Transduktion mittels konfokaler Mikroskopie Spinning-Disk-

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

live-cell-imaging-primary-rat-neonatal-cardiomyocytes-following

Research

JoVE Journal

Biology

13.5K Aufrufe.  Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology. This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Serie: Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Isolierung und Kryokonservierung von neonatalen Rattenkardiomyozyten

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Forschung

Entwicklungsbiologie

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualisierung von Zellzyklus-Variationen und Bestimmung der Nukleation in postnatale Kardiomyozyten

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Live-Imaging von Single Cell Divisionen in Maus Neuroepithel

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Neurowissenschaften

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Isolation und genetische Manipulation von Adult Herzmuskelzellen für konfokale Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Biologie

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Ultraschall-geführte Transthorakale intramyokardiale Injection in Mäuse

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Medizin

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Isolierung und Kultivierung von Erwachsenen Ratte Kardiomyozyten

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Isolation und Kultur der Neugeborenen-Maus-Kardiomyozyten

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Zeitraffer-Bildgebung neuronaler Arborisation mittels spärlicher adenoassoziierter Virusmarkierung genetisch zielgerichteter Netzhautzellpopulationen

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolierung, Kultur und Transduction von erwachsenen Maus Kardiomyozyten

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Analyse der kardialen kontraktilen Dysfunktion und Ca2+ Transienten in Nagetiermyozyten

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...