We like to acknowledge Marcelo Toro for the assistance with flow cytometry. This study was supported by Swedish Heart- and Lung Foundation, EU Commission FP7 "CardioCell", Swedish Research Council, AFA insurances and ALF. O.B. was supported by Deutsche Forschungsgemeinschaft.

1. Isolation of the Cardiac Nuclei
<ol>
 <li>Coat ultracentrifuge tubes (Beckman Centrifuge Tubes #363664) with 10 ml of 1%BSA/PBS coating solution. Cap the tubes and let them rotate for 30 min in a tube rotator. Remove the coating solution and let the centrifuge tubes air dry (one tube per mouse heart is required for the analysis of single mouse hearts, alternately up to 5 mouse hearts or 1 g of heart tissue from a different species (e.g. human) can be processed in one tube).</li>
 <li>All following steps should be pe...

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. 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Vis. Exp. (20), e914 (2008).</li></ol>

Accurate identification of cardiomyocyte nuclei is crucial for the analysis of regenerative processes in the myocardium2,3. Conventional techniques to isolate cardiomyocytes from fresh tissue are mainly based on enzymatic digestion of extracellular matrix proteins and the subsequent purification from interstitial cells by low speed centrifugation. Further purification of living cardiomyocytes from embryonic stem cells (ESC) can be performed by immunolabeling with surface markers such as SIRPA9 or mi...

<table border="1">
 <tbody>
 <tr>
 <td>1. Lysis Buffer</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>0.32 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 8)</td>
 </tr>
 <tr>
 <td>5 mM CaCl2</td>
 </tr>
 <tr>
 <td>5 mM magnesium acetate</td>
 </tr>
 <tr>
 <td>2.0 mM EDTA</td>
 </tr>
 <tr>
 <td>0.5 mM EGTA</td>
 </tr>
 <tr>
 <td>1 mM DTT</td>
 </tr>
 </tbody>
</table>
 
<table border="1">
 <tbody>
 <tr>
 <td>2. Sucrose buffer</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>2.1 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 8)</td>
 </tr>
 <tr>
 <td>5 mM magnesium acetate</td>
 </tr>
 <tr>
 <td>1 mM DTT</td>
 </tr>
 </tbody>
</table>
 
<table border="1">
 <tbody>
 <tr>
 <td>3. Nuclei storage buffer (NSB plus)</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>0.44 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 7.2)</td>
 </tr>
 <tr>
 <td>70 mM KCl</td>
 </tr>
 <tr>
 <td>10 mM MgCl2</td>
 </tr>
 <tr>
 <td>1.5 mM spermine</td>
 </tr>
 </tbody>
</table>

<table border="1">
 <tbody>
 <tr>
 <td>Reagents and Equipment</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>Isotype rabbit IgG- ChIP Grade, #ab37415</td>
 <td>Abcam</td>
 </tr>
 <tr>
 <td>Rabbit anti-PCM-1 antibody, #HPA023374</td>
 <td>Atlas Antibodies</td>
 </tr>
 <tr>
 <td>Donkey sec. antibody, anti-rabbit Alexa 488 Fluor, #A-21206 or equivalent sec. fluorescent antibody</td>
 <td>Life Technologies</td>
 </tr>
 <tr>
 <td>DRAQ5</td>
 <td>Biostatus</td>
 </tr>
 <tr>
 <td>cell strainers 30 &mu;m, 70 &mu;m and 100 &mu;m</td>
 <td>BD Biosciences</td>
 </tr>
 <tr>
 <td>Glass douncer (40 ml) and pestle "L"</td>
 <td>VWR (Wheaton Industries Inc.)</td>
 </tr>
 <tr>
 <td>T-25 Ultra-Turrax Homogenizer</td>
 <td>IKA Germany</td>
 </tr>
 <tr>
 <td>Dispersing tool S25 N-18 G</td>
 <td>IKA Germany</td>
 </tr>
 <tr>
 <td>Beckman Avanti Centrifuge</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Falcon Tubes 15 ml and 50 ml</td>
 <td>VWR</td>
 </tr>
 <tr>
 <td>Beckman Centrifuge Tubes #363664</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>JS13.1 free swinging rotor</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Influx cytometer</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Tube Rotator</td>
 <td>VWR</td>
 </tr>
 </tbody>
</table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Research

JoVE Journal

Medicine

16.8K Views.  University of Lund. Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.