This work was supported by the German National Science Foundation (DFG) and by the Federal Institute for Risk Assessment (BfR, Germany).

The over all design of the protocol containing four major steps is depicted in Figure 1. All steps are described in full detail. Cell isolation, transduction and culture, followed by confocal imaging about 24 hours later is a consecutive and compulsory timescale. The virus construction should be done ahead of the cell isolation and is then used for the genetic transduction.
 1. Isolation of adult cardiac myocytes from rat heart
 <ol>
 <li>Adult male Wistar rats approximately 250 g in weight (6-12 weeks old) are anesthetised by intraperitoneal injection of 170 &mu;l anaesthesia solution per 100 g bodyweight</li>
<li>Additionally, a citrate solution (2 &mu;l / g body weight) is injected to avoid blood coagulation during the surgery.</li>
<li>Langendorff Perfusion Set-Up should be prepared. All solutions should be saturated with oxygen. </li>
<li>When the rat is anesthetised, the animal is fixed on a surgery board and the upper part of the body is disinfected with 70% ethanol.</li>
<li>The animal is killed by cutting through the carotid.</li>
<li>The Thorax is opened using scissors. The heart and the aorta should now be accessible. The pericard is removed.</li>
<li>Ice-cold solution A+ (5 ml) is injected in both ventricles using a 26-gauge needle to arrest the heart and wash out blood.</li>
<li>The aorta is grabbed with a clamp and truncated at the aortic arch just were the first branching occurs.</li>
<li>The heart is removed from the chest and the aorta is attached to the cannula of a perfusion set-up by forceps and a clamp to allow retrograde Langendorff-perfusion. Be aware not to foraminate the aortic valves.</li>
<li>The heart needs to be fixed by a ligature around the aorta/cannula. Open vessels also need to be ligated.</li>
<li>The heart is retrogradely perfused with oxygen-saturated solution A+ at room temperature (23&deg;C) for 10 min at a pressure of approximately 70 mbar. Make sure no air bubbles enter the aorta.</li>
<li>The perfusion solution is switched to a Liberase solution at a temperature of 37&deg;C for approximately 25 min at a rate of 4 ml/min by means of a peristaltic pump. At the end of the perfusion the heart should look marbled white. The digestion time may need to be adapted to the situation/tissue condition.</li>
<li>The heart is taken from the perfusion set-up and placed in a Petri dish containing solution A at a temperature of 37&deg;C. Remaining vessels and atria are cut off. The vessels are discarded and the atria - if needed - are treated differently (not part of this protocol). The ventricles are devided into approximately 6 pieces.</li>
<li>The pieces are transferred into a 100 ml Erlenmeyer flask containing 20 ml solution A at a temperature of 37&deg;C and slightly agitated in a 37&deg;C water bath for 5 min. Finally the supernatant is discarded.</li>
<li>The procedure described above is repeated once or twice. The supernatant should appear slightly opaque.</li>
<li>The increment of the extracellular calcium concentration is initiated by adding 20 ml of solution B1/2 at 37&deg;C to the cells and tissue remaining a residual of solution A in the Erlenmeyer flask. This is slightly shaken in a 37&deg;C water bath for an additional 5 min. Subsequently the supernatant is discarded.</li>
<li>Add further 20 ml solution B1/2. (The solution/supernatant should now contain viable cells.) A drop of the cells is transferred to a petri dish on an inverted cell culture microscope. If the cells show a high spontaneous activity (contractions) one needs to wait until cells come to rest.</li>
<li>Once more the supernatant is discarded and 20 ml of solution B1/2 are added.</li>
<li>The tip of a plastic Pasteur pipette is cut off in such a way that the tissue pieces can pass easily when agitated through the opening. With flame treatment sharp edges are softened (melted).</li>
<li>Now the tissue pieces are slightly and gently triturated. The tissue pieces should "fall apart" into the cells.</li>
<li>The cell containing supernatant is decanted and diluted with solution B to reach the desired cell density, which depends on the type of experiments and should be determined empirically.</li>
</ol>
2. Construction of an adenovirus for fusion protein transduction
To produce adenoviruses the Transpose-Ad Adenoviral Vector System from MP Biomedicals is used1. This protocol starts with the available and tested entry vector (pCR259) for a fluorescent fusion protein or GEB s2.
<ol>
<li>An amount of 10 ng of the above mentioned initial pCR259 vector containing the gene of interest is transformed with 100 &mu;l HighQ-1 Transpose-AD&trade; 294 competent cells.</li>
<li>This mixture is incubated for 20 min on ice followed by a heat shock for 45 s at 42&deg;C and stored on ice for an additional 2 min. </li>
<li>Add 900 &mu;l of unselective LB-medium and grow at 37&deg;C with gentle agitation for 4 hours.</li>
<li>Plate 50 &mu;l, 100 &mu;l and 200 &mu;l on plates containing LB-medium with Chloramphenicol (Cm, 20 &mu;g/ml), Ampicillin (100 &mu;g/ml), tetracycline (15 &mu;g/ml), X-Gal (280 &mu;g/ml) and IPTG (40 &mu;g/ml). (A more intense and faster blue staining is possible by using the more expensive dye Bluo-Gal with 300&mu;g/ml. This should be used in particular when you are not so familiar in recognizing and distinguishing blue/white colonies)</li>
<li>Grow for 24 hours at 37&deg;C. Leave at 4&deg;C until good identification of blue colonies is possible. When there are problems to decide which colony is truly white pick some of the colonies and replicate on another plate containing the three antibiotics, X-Gal and IPTG and let them grow over night at 37&deg;C. 
 Remark) The HighQ-1 Transpose-AD&trade; 294 cells contain two important plasmids: The adenovirus backbone (Transpose-Ad&trade; 294) and a helper plasmid expressing a transposase, which mediates the insertion of the gene of interest into its target sites, located in the LacZ gene of the Transpose-AD 294 plasmid.</li>
<li>To identify positive colonies containing the gene of interest in the virus backbone perform colony-PCR as described in 2.7-2.9. Use vector primers flanking the multiple cloning site (forward 5'CCCAGTCACGACGTTGTAAAACG3', reverse 5'GCGGATAACAATTTCACACAGG3') and make a colony PCR with 100% white colonies and use one blue colony as a control for Transpose-AD 294 plasmids without inserted gene of interest. Blue colonies will give you a short PCR fragment (~200bp), colonies containing your gene of interest show a large PCR product (depending on the length of your gene of interest) and colonies with two fragments are multiple cell colonies and have to be separated if further used.</li>
<li>According to the table below a master mix needs to be prepared.</li>
<li>Pick a single 100% white colony with an autoclaved toothpick and first make a replicate of this colony on a LB-plate with Cm.</li>
<li>Transfer the colony into the PCR-mix. Twirl it in the PCR-mix 3-4 times than remove the toothpick before it could soak a lot of the PCR-mix. The PCR-protocol depends on the kb of the insert and the used polymerase. The annealing temperature of the primers is 57&deg;C and a use of 35 cycles is recommended.</li>
<li>Grow over night cultures (LB-medium with Cm) of the positive clones and isolate the plasmids with a commercial available kit without spin columns, because the centrifugal force disrupts the huge virus vector.</li>
<li>Transform 0.5 &mu;g of the isolated plasmids in 100 &mu;l DH5&alpha; competent cells. Incubate DNA with cells on ice for 30 min. Heat shock 45 s at 42&deg;C and set on ice for 2 min.</li>
<li>Add 900 &mu;l unselective LB-medium and grow at 37&deg;C with gentle agitation for 1 hour.</li>
<li>Plate 50 &mu;l on a plate containing LB-medium with Cm, X-Gal and IPTG. Incubate at 37&deg;C for 24 hours.</li>
<li>Pick and replicate up to 20 100% white colonies on separate Ampicillin, Tetracycline and Chloramphenicol plates containing IPTG and X-Gal. Let them grow over night at 37&deg;C. Select colonies which are 100% white, chloramphenicol resistant, ampicillin and tetracyclin sensitive. </li>
<li>Colony-PCR (optional): positive clones can be tested again with colony-PCR to check that they contain the insert.</li>
<li>Pick a single colony of a positive recombinant and grow in 600 ml LB-medium with Cm.</li>
<li>Isolate the plasmid using a plasmid maxi kit.</li>
<li>Digest 5 &mu;g of plasmid with Pac I for two hours at 37&deg;C and stop the reaction at 65&deg;C for 20 min.</li>
<li>Concentrate the whole digest in a SpeedVac at room temperature for 30 min to a final volume of 10 &mu;l or use a phenol-chloroform extraction method.</li>
<li>Thaw a vial of frozen QBI-HEK 293 cells with gentle agitation in a 37&deg;C water bath. Rinse the outside of the vial with 70% ethanol and proceed aseptically in a sterile hood.</li>
<li>Transfer cells to a sterile 15 ml tube, add dropwise 10 ml DMEM (+10% FCS) while gentle agitating and centrifuge 10 min at 250xg to pellet the cells.</li>
<li>Discard supernatant. Resuspend the pellet in 10 ml DMEM (+10% FCS) by gently pipetting up and down.</li>
<li>Seed in a 75 cm2 flask and add 10 ml DMEM with 10% FCS to the cells. Incubate over night at 37&deg;C in a 5% CO2 incubator.</li>
<li>The next day verify the cell density. QBI-HEK 293 cells should be split at a 1:10 to 1:20 ratio.</li>
<li>Remove the medium from the cells and trypsinize with 2 ml Trypsin for 1-3 min. Give 10 ml DMEM to the cells and centrifuge at 250xg for 10 min. </li>
<li>Remove carefully the medium and give 10 ml fresh medium to the cells, resuspend and count the cells.</li>
<li>Plate in a six-well one well with 2x105, 3x105, 4x105, 5x105 and 6x105 cells. Incubate the dish at 37&deg;C in a CO2-incubator.</li>
<li>At the following day use a well with 50% confluence to transfect with the concentrated Pac I digest. We have good experience with Lipofectamine 2000 transfection, but also other methods (such as calcium phosphate transfection) should be suitable.</li>
<li>Change the medium in the well 3-4 h before transfection to get the cells into exponential growth during the procedure.</li>
<li>Mix 5 &mu;l Lipofectamine with 250 &mu;l DMEM and incubate at room temperature for 5 min.</li>
<li>Give the Lipofectamine-Medium Mixture to the DNA (Pac I digest) and mix by tapping against the reaction tube, do not mixing by pipetting. Incubate the mixture 20 min at room temperature.</li>
<li>Drop the transfection mixture to the cells and rock the plate to mix. Incubate at 37&deg;C with 5% CO2. Change medium after 4-5 hours. </li>
<li>Two days after transfection take the medium from the cells and give it into a fresh culture flask (75 cm2).</li>
<li>Trypsinize the cells with 500 &mu;l trypsin for up to 5 min. Give 1 ml DMEM to the cells, take the whole mixture and put it, together with 17 ml fresh medium (DMEM with FCS) into the culture flask. Cultivate the cells at 37&deg;C with 5%CO2 until cytopathic effects (CPE) are visible. See Figure 2 to learn how CPE should look like.</li>
<li>Collect the cells with the medium in a 50 ml tube and freeze the cells at -80&deg;C. Break the cells with three freeze/thaw cycles (-80&deg;C/37&deg;C water bath).</li>
<li>Centrifuge 20 min at 8000xg and 4&deg;C. Decant the supernatant into a 100mm-diameter dish and discard the pellet. Use a sterile filter with 0.45&mu;m pore size to filtrate the supernatant. This is the first virus stock (passage 1). </li>
<li>Use 1/3 of this stock to transduce a 75cm2 flask with QBI-HEK 293 cells with 70% confluence. Incubate at 37&deg;C and 5%CO2 until CPE s are visible. </li>
<li>If the cells show CPE s collect them and extract the viruses as done before. This is passage 2 of the virus.</li>
<li>Repeat steps 2.37 and 2.38 with virus passage 2 and so on until the amplification of the viruses reaches a sufficient virus stock. The virus can be concentrated with specific centrifugation tubes (Amicon Ultra-15 Centrifugal filter unit with ultracel-100 membrane from Millipore) and stored for several months at -80&deg;C. To count the infectious particles use a plaque assay.</li>
<li>Use virus passage 2 or higher to transduce cells and for further amplification of the virus.</li>
</ol>
3. Primary cell culture and adenoviral transduction
<ol>
<li>Sterile round cover slips of 20 mm diameter are placed in standard 12-well plates.</li>
<li>20 &mu;l ECM solution is equally distributed on each cover slip. Remains are discarded. For drying allow a minimum of 1 h at 37&deg; C. Alternatively the coating can by dried over night (12 h) at room temperature. </li>
<li>Each well is filled with 1 ml Medium M199 with supplements comprising antibiotics and ITS.</li>
<li>A cell suspension (from step 1.21) of 200 to 400 &mu;l is added to each well. Culturing of the cells occurs at 37&deg;C in a 5% CO2 incubator. </li>
<li>Cells are allowed to sediment and after 1 h medium is changed. </li>
<li>Immediately after changing the medium an adequate amount of virus containing solution (from passage 2 or higher) is mixed by gently rocking the plate.</li>
<li>In case of a long term culture medium is changed every 2 days.</li>
<li>24 hours after transfection GEB expression is detectable.</li>
</ol>
4. Confocal imaging for 3D subcellular structures and Ca2+ Signaling
Confocal imaging can be performed on any confocal microscope. However, calcium-imaging experiments require an acquisition speed of at least video rate (25-30 Hz). This restricts usable technologies to either multi beam scanners (e.g. Nipkow disc, swept field or kilo-beam array scanner) or the very fast single beam scanner (e.g. resonant scanner or acousto optical deflector (AOD)-scanner). For an overview see Ref. 3. The 3D-subcellular imaging requires a z-drive. Here a kilo-beam array scanner (VTinfinity, VisiTech Int., Sunderland, UK) is used.
<ol>
<li>The microscope and peripheral equipment needs to be set into an operation mode. Especially for the laser this may require a warming up period until a stable output power is reached.</li>
<li>Cells need to be transferred to an experimental chamber (cover-slip transfer). The coverslips with cells are transferred to an experimental chamber. 1mL Tyrode solution (containing 1.8 mM extracellular calcium) is then added.</li>
<li>The experimental chamber needs to be placed on the microscope stage. Stimulation electrodes, perfusion set-up and further peripheral devices, e.g. temperature control, need to be arranged accordingly.</li>
<li>Cells will be focused at and selected using white light and/or epifluorescent illumination.</li>
<li>Acquisition parameters such as laser power, exposure time etc. or an entire experiment protocol needs to be set-up.</li>
<li>Time series and/or Z-section images can be acquired.</li>
<li>On the set-up further advanced multimode measurements can be done using electrophysiology (patch-clamp) or/and optical manipulation like fluorescence redistribution after photobleach (FRAP) or uncaging of substances including two photon photolysis.</li>
<li>Image analysis, especially 3D-reconstruction is performed on dedicated analysis software such as Imaris (Bitplane AG, Z&uuml;rich, Switzerland), VeloCity (Improvision, Coventry, UK) or Arivis Browser (arivis GmbH, Rostock, Germany).</li>
</ol>
5. Representative Results:
The final outcomes of the protocol are image sequences that can be used to extract further data.
An example is the investigation of subcellular structures and organelles. Figure 3 depicts the example of the 3-dimensional arrangement of the Golgi apparatus. In contrast to electron microscopy all investigations can be performed on living cells.
<img src="/files/ftp_upload/1433/JoVEFig1.jpg" alt="Figure 1" /> Figure 1: General flow of the major experimental steps.
<img src="/files/ftp_upload/1433/JoVEFig2.jpg" alt="Figure 2" /> Figure 2: The left image depicts the Q-HEK cells in culture with 90-95% confluency which are not transduced. Cytopatic effects after viral transduction are shown in the right part. Cells round up and detach from the bottom of the plate. In these cells the nucleus occupies the major part of the cells as a result of active virus production. 
<img src="/files/ftp_upload/1433/JoVEFig3.jpg" alt="Figure 3" /> Figure 3: Adult rat cardiac myocyte transfected with an YFP-fusion protein targeting the Golgi apparatus. Cardiac myocytes after adenoviral gene transfer (one day in culture) express the fluorescent fusion protein. Panel A depicts a single confocal slice taken from a z-stack. In panel B the same cell is depicted after deconvolution using a theoretical point spread function (PSF). This de-blurred image stack is subsequently used to render the cell and get a 3D volume reconstruction as shown in panel C or a 3D surface reconstruction (D). The bar represents 10 &mu;m.

<ol>
	<li>Kaestner, L., Ruppenthal, S., S, ., Licha, K., Lin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Imaging II. Vol. 7370, 737008-737008 (2009).</li><li>Viero, C., Kraushaar, U., Ruppenthal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Cell Calcium. 43 (1), 59-59 (2008).</li><li>Kaestner, L., Lipp, P., Shorte, S. L., Frischknecht, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Cellular and Molecular Biological Functions. , 289-289 (2007).</li><li>Kaestner, L., Lipp, P., Popp, J., von Bally, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optics in Life Science. Vol. 6633, 66330K-66330K (2007).</li><li>Rinne, A., Littwitz, C., Bender, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+delivery+of+short+hairpin+RNA+(shRNA)+mediates+efficient+gene+silencing+in+terminally+differentiated+cardiac+myocytes.">Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.</a> Methods Mol Biol. 515, 107-107 (2009).</li></ol>

The procedure described for the isolation of rat cardiomyocytes can be adapted to other species, e.g. mouse4, if necessary. A parameter that needs adaptation is the enzyme mix for digestion (see below) and also the duration of digestion. Be aware that the cells of some species (e.g. mouse) might be more fragile than others (e.g. rat).
The choice of collagenase is probably the most crucial step in the isolation procedure. We choose Liberase Blendzyme because it is a synthetic enzyma...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anesthesia solution</td>
<td>&nbsp;</td>
<td>Serumwerk Bernburg GmbH (Ursotamin), Bayer Health Care (Rompun)</td>
<td>&nbsp;</td>
<td>Mixture of 1 ml Ursotamin (100 mg/ml Ketaminhydrochlorid) and 240 &mu;l Rompun (2% Xylazinhydrochlorid)</td>
</tr>
<tr>
<td>Citrate solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>117,64 mg sodium citrate in 10 ml 0.9% NaCl solution</td>
</tr>
<tr>
<td>Solution A</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>134 mM NaCl, 4 mM KCl, 11 mM glucose, 1.2 mM MgSO4, 1.2 mM Na2HPO4, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Solution A+</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 0.2 mM EGTA, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Liberase solution</td>
<td>&nbsp;</td>
<td>F. Hoffmann&#x2010; La Roche Ltd.</td>
<td>11988476 001</td>
<td>500 &mu;l stock solution in 15 ml solution A (stock solution:
10 mg/ml Liberase Blendzyme 4 in aqua dest.)</td>
</tr>
<tr>
<td>Solution B</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Content of solution A plus 200 &mu;M CaCl2 and 0.1% DNase solution</td>
</tr>
<tr>
<td>Solution B1/2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1:1 mixture of solution A and solution B</td>
</tr>
<tr>
<td>DNase solution</td>
<td>&nbsp;</td>
<td>Sigma&#x2010; Aldrich Co.</td>
<td>D4527</td>
<td>Deoxyribonuclease I, Type 2 from bovine pancreas, 40 KU (15 mg) are dissolved in 5 ml of 10 mM Tris&#x2010;HCl buffer, adjusted to pH=7.35, additional content: 50 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol; this solution is mixed with 5 ml glycerol</td>
</tr>
<tr>
<td>Extracellular matrix protein (ECM) solution</td>
<td>&nbsp;</td>
<td>Extracellular matrix protein (ECM) solution</td>
<td>E1270</td>
<td>1 ml stock solution as purchased is diluted with 6 mL
medium M199</td>
</tr>
<tr>
<td>Culture medium M199</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>E15&#x2010;834</td>
<td>Medium M199 supplemented with 1 &mu;l/ml ITS solution and 20 &mu;l/ml penicillin&#x2010;streptomycin solution (5000 units/ml)</td>
</tr>
<tr>
<td>ITS solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>25 mg Insulin, 25 mg Transferrin and 50 &#x03BC;l Selenite stock solution are dissolved in 5 ml aqua dest. (stock solution: 0.5 mg/ml Sodiumselenite in aqua dest.). Add a few drops of 1 M HCl until one should get a clear
solution. Aliquots can be frozen.</td>
</tr>
<tr>
<td>Tyrode</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 2mM MgCl2, 10 mM glucose, 10 mM HEPES, pH adjusted to 7.35 using 10 M NaOH, sterile filtered</td>
</tr>
<tr>
<td>Master mix</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>5 &mu;l 10&#215;PCR&#x2010;Buffer, 5 &mu;l MgCl2 (25 mM), 2 &mu;l forward primer, 2 &mu;l reverse primer, 1 &mu;l dNTP&#180;s (10 mM each), 33 &mu;l H2O, 1 &mu;l DMSO, 1 &mu;l Taq&#x2010;DNA polymerase for each sample</td>
</tr>
<tr>
<td>LB&#x2010;Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 g/l Tryptone, 5 g/l Yeast extract, 5 g/l NaCl (and 15 g/l Agar if used as solid medium), pH 7.0</td>
</tr>
<tr>
<td>Pac I</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>R0547L</td>
<td>preparation: 50 &mu;g DNA, 5 &mu;l 10&#215;buffer, 0.5 &mu;l 100&#215;BSA,
0.75 &mu;l Pac I, fill with H2O up to final volume of 50&mu;l</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging at JoVE.com

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

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 ...

isolation-genetic-manipulation-adult-cardiac-myocytes-for-confocal

Research

JoVE Journal

Biology

14.5K Aufrufe.  Universty of Saarland. Adult Herzmuskelzellen sind primäre Zellen, die aus tierischen Herzen isoliert und kultiviert für mehrere Tage. Innerhalb dieses Zeitraums Kultur adenoviralen Gentransfer können verwendet werden, um genetisch kodierte Biosensoren (GEBS) oder fluoreszierende Fusionsproteine ​​zu exprimieren. Beide Ansätze erlauben zellulären Untersuchungen mittels der konfokalen Mikroskopie.

Serie: Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...