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.

Primary cardiomyocytes isolated from neonatal rats have long been used to study cardiomyocyte functions in vitro. This protocol describes a method for the isolation of neonatal cardiomyocytes from rat pups using a two-step enzyme digestion method, first digesting with trypsin O/N at 4 &#176;C and then purified collagenase. One advantage of employing the purified collagenase step is that the same grade of enzyme is used for all isolations. Thus, the quality and amount of isolated cells is consistent from experime...

Primary rat neonatal cardiomyocytes have long been used for investigating cardiomyocyte function in vitro1. They are easy to isolate from rat pups by several well established methods2-4. The most common method employs collagenase or trypsin to digest connective tissue of the heart prior to cell isolation. Researchers have also developed methods for isolating cardiomyocytes from adult rodents5-8 as well as neonatal mice9,10.
This protocol describes a method for isolating cardiomyocytes from neonatal rat pups, employing a two-step enzyme digestion procedure. Trypsin is first used O/N at 4&#160;&#176;C, and then purified collagenase at 37&#160;&#176;C. Incubation of the heart tissue with trypsin O/N at 4&#160;&#176;C&#160;reduces the steps necessary to harvest cells compared to methods using sequential incubations in a warm enzyme solution2. In addition, by using purified collagenase rather than crude enzymes, lot-to-lot variability can be eliminated, thus providing enhanced reproducibility.
Functional studies of a particular protein often employ a protein expression system using an adenovirus11-13 and/or a lentivirus14-16. [CAUTIONARY NOTE] The viral production and manipulation should be carried out according to the NIH guidelines.
The adenovirus does not integrate into the host genome. It has a very high efficiency of transduction in most cell types, including dividing cells and non-dividing cells, as well as primary cells and established cell lines. This makes the adenovirus a reliable vector for gene expression. High levels of the protein encoded by the adenovirus vector develop within 48 hr following transduction, and they can last for several weeks17. However, one drawback to using an adenovirus for protein expression is that the development of a recombinant adenovirus is both complicated and time consuming. This drawback explains in part why many researchers have been turning to lentiviruses for recombinant gene expression. Unlike adenoviral constructs, generating a lentiviral construct is quick and easy. Although lentiviruses generally have lower efficiencies of transduction than adenoviruses, in both dividing and non-dividing cells, they do integrate into the host genome. Consequently, expression of the transduced gene is more stable for lentiviruses than for adenoviruses.
Due to technological advances in the field of microscopy, it is much easier to capture live cell images of cells expressing recombinant proteins. This holds true even at video rate speeds of acquisition. This allows the investigator to determine how particular alterations in the protein of interest functionally impact the cell in real time. The confocal spinning disk microscope has several key features that make it an optimal technique for live cell imaging18,19. The Yokogawa spinning disc allows for much more rapid image acquisition, while at the same time utilizing far less laser power than point scanning confocal microscopes. Both of these special features are due to the spinning disk, which contains numerous confocal holes through which the laser passes simultaneously to the samples. During acquisition, the disk itself spins quickly and continuously18-20. By using the autofocus system of the microscope, stable focus is maintained over long periods of time. This allows researchers to take well-focused live cell images. Acquired images are played back as a movie file. The images are analyzed using image analysis software such as ImageJ21,22, FIJI23, or other commercially available software.

<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

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

To illustrate the technique, a lentivirus encoding EGFP (enhanced green fluorescent protein)-tagged Cx43 (Connexin43) or a mutated Cx43 32 was used to express EGFP-tagged proteins in cells, and an adenovirus encoding FGFR1DN (fibroblast growth factor receptor 1 dominant negative) was used to shut down FGF signaling in the cell 33-35. Three days following the isolation of cardiomyocytes, the isolated cardiomyocytes were transduced with lentivirus in order to express EGFP-tagged proteins in the cells....

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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.

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

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Research

JoVE Journal

Biology

13.5K Views.  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.

Video: 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.

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

Developmental Biology

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.

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

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

Neuroscience

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.

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 ...

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.

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

Medicine

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.

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

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Bioengineering

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.

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.

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.

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

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

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