Name: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue (Video) | JoVE Journal
Uploaded: 2012-07-10T13:00:00.000+00:00
Duration: 10 min 12 s
Description: Learn to isolate cardiomyocyte nuclei from postmortem tissue using homogenization, density sedimentation centrifugation, and fluorescence activated cell sorting with PCM-1 immunolabeling. This method achieves high purity for downstream cardiac research.

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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 performed on ice. Dissect the left ventricle from fresh or snap-frozen mouse heart with a scalpel. Note, this protocol is optimized for mouse heart but can also be adapted to rat or human heart. Alternatively, use up to 1 g of heart tissue from a different species.</li>
 <li>Trim the specimen into small cubicles with a scalpel.</li>
 <li>Transfer the tissue pieces into a 50 ml Falcon tube filled with 15 ml of lysis buffer.</li>
 <li>Homogenize the heart tissue with a T-25 Ultra-Turrax probe homogenizer (IKA) at 24,000 rpm for 10 sec.</li>
 <li>Dilute the homogenate with equal volume of lysis buffer to 30 ml.</li>
 <li>Use a glass douncer (40 ml) to further homogenize the tissue and free the nuclei. Perform eight strokes with a large clearance pestle.</li>
 <li>Pass the crude nuclei isolate through a 100 μm and 70 μm nylon mesh cell strainer (BD Biosciences), consecutively.</li>
 <li>Spin down the crude nuclei isolate in a refrigerated centrifuge (4 °C) at 700 x g for 10 min.</li>
 <li>Remove the supernatant carefully by inverting the tubes and wipe the inside of the tube with paper towel. Be careful not to disturb the nuclei pellet.</li>
 <li>Dissolve the crude nuclei isolate in 5 ml of sucrose buffer by pipetting the solution several times up and down. Add a further 25 ml of sucrose buffer to the dissolved pellet.</li>
 <li>Add 10 ml of freshly prepared sucrose buffer to the coated ultracentrifuge tube (see step 1.1).</li>
 <li>Carefully overlay the added 10 ml of sucrose buffer with the resuspended nuclei pellet dissolved in sucrose buffer from step 1.9.</li>
 <li>Balance the centrifuge tubes before placing them into a JS13.1 free swinging rotor and place the rotor into a high-speed centrifuge (Beckman Avanti S-25).</li>
 <li>Spin the nuclei sample at 13,000 x g at 4 °C for 60 min.</li>
 <li>When the spin has completed, remove the tubes carefully from the rotor and discard the supernatant by inverting the tubes and wiping the remaining debris from the inside of the tubes with paper towel.</li>
 <li>Dissolve the nuclei pellet in 1 ml of nuclei storage buffer (NSB plus buffer). Note: NSB plus contains 1.5 mM spermine as a DNA stabilizer.</li>
 <li>Proceed with step 2.1, immunostaining of cardiomyocyte nuclei.</li>
</ol>
2. Immunostaining for Flow Cytometry
<ol>
 <li>Prepare the negative control for immunostaining. Take an aliquot of 20 μl out of the nuclei sample and add 980 μl of NSB plus buffer.</li>
 <li>Add anti-pericentriolar material 1 antibody (rabbit anti-PCM-1, Atlas Antibodies) to the nuclei sample in a dilution of 1:500 to immunolabel cardiomyocyte nuclei. Add the isotype antibody in the same dilution as the anti-PCM-1 antibody to the negative control, prepared in step 2.1.</li>
 <li>Incubate negative control and sample tube at 4 °C overnight.</li>
 <li>Wash negative control and sample at least once with NSB plus buffer (spin down tubes in a refrigerated centrifuge (4 °C) at 700 x g for 10 min. Discard supernatant and dissolve the nuclei pellet in 1 ml of NSB plus buffer).</li>
 <li>Add anti-rabbit fluorescent secondary antibody (FITC or APC) to negative control and sample tube in a dilution of 1:1000.</li>
 <li>Incubate negative control and sample tube at 4 °C for 1 h.</li>
 <li>Wash negative control and sample at least once with NSB plus buffer (spin down tubes in a refrigerated centrifuge (4 °C) at 700 x g for 10 min. Discard supernatant and dissolve the nuclei pellet in 1 ml of NSB plus buffer).</li>
 <li>Proceed with the flow cytometric analysis and sorting.</li>
</ol>
3. Flow Cytometry
<ol>
 <li>Coat nuclei collection tubes (Falcon 15 ml) with 1%BSA/PBS solution before you start flow cytometry sorting as described in step 1.1.</li>
 <li>Filter the sample and the negative control through a 30 μm cell strainer and load first the negative control to the flow cytometer (BD Influx). Define the first and second gate to define nuclei and singlets (single nuclei), based on forward scatter (FSC), forward scatter pulse width (FS pulse width) and side scatter (SSC) (Fig. 2a and b). Adding a DNA stain (DRAQ5 (1:500)) to the sample can help to identify the nuclei population initially.</li>
 <li>Load the immunolabeled sample and define the third gate to isolate cardiomyocyte nuclei (PCM-1-positve) from non-cardiomyocyte nuclei (PCM-1-negative). Start the sorting (Fig. 2c and d).</li>
</ol>
Optional: In order to analyze the nuclear DNA content (ploidy) and to perform cell cycle analysis add an appropriate DNA stain to the nuclei (e.g. Hoechst 33342 or DRAQ5) (Fig. 2e).
<ol start="4">
 <li>After flow cytometry sorting, place the nuclei on ice and re-analyze to determine the sorting purity (Fig. 3a and b).</li>
 <li>Spin down the sorted nuclei in the collection tubes at 1500 x g in a refrigerated centrifuge for 15 min.</li>
 <li>Dissolve the nuclei pellet in a buffer compatible with the downstream application.</li>
</ol>
4. Representative Results
Nuclei morphology and integrity can be assessed by DNA stains and visualized by microscopy (Fig. 1). Successful PCM-1 labeling can be assessed by epifluorescence microscopy and by flow cytometry (Fig. 1 and Fig. 2c and d). PCM-1-positive and negative populations should be well separated from each other (Fig. 2c and d). In murine left ventricle about 30% of all nuclei should be cardiomyocyte nuclei (Fig. 2d). Sorting purity can be assessed by re-analyzing the sorted nuclei (Fig. 3a and b). Both nuclei populations should have a sorting purity exceeding 95%.
<img alt="Fluorescence microscopy, protein expression analysis; nuclei, PCM-1, Nkx2.5 markers, cellular study." src="/files/ftp_upload/4205/4205fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. PCM-1 identifies cardiomyocyte nuclei. Cardiac nuclei (a) are stained with antibodies to PCM-1 (b) and to Nkx2.5 (c) in an adult mouse heart. (d) PCM-1-labeled nuclei are surrounded by cardiomyocyte cytoplasm (myosin heavy chain (MHC)) and express the transcription factor Nkx2.5, documenting the accurate identification of cardiomyocyte nuclei by PCM-1 staining (scale bars 20 μm and 10 μm (d, inset)). (e) Cardiac nuclei isolates visualized with the DNA stain DRAQ5. (f and g) Cardiomyocyte nuclei are labeled with antibodies against PCM-1 (scale bar 10 μm). Note, the epinuclear staining pattern of PCM-1 in myocyte nuclei in tissue section and in isolated nuclei (arrows).
<img alt="Flow cytometry analysis; scatter plots illustrating cell populations; DNA content histogram." src="/files/ftp_upload/4205/4205fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Flow cytometric sorting of cardiomyocyte nuclei. (a) Cardiac nuclei are identified by forward scatter (FSC) and side scatter (SSC). (b) A second gate identifies single nuclei by FSC and FS pulse width5. (c, d) Fluorescent gating allows the separation of cardiomyocyte nuclei (PCM-1-positive) and non-cardiomyocyte (PCM-1-negative) nuclei from heart tissue. (e) Mouse cardiomyocyte are mostly (&gt;80%) diploid (2n), only a small subset is tetraploid (4n)6. Note, human cardiomyocytes contain a higher frequency of polyploidy nuclei (&gt;2n)7,8.
<img alt="Flow cytometry scatter plot comparison; PCM-1 vs SSC; cell population analysis diagram." src="/files/ftp_upload/4205/4205fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Purity analysis of sorted cardiomyocyte and non-cardiomyocyte nuclei. Re-analysis of sorted non-cardiomyocyte (a) and cardiomyocyte nuclei (b). Both populations show a sorting purity exceeding 99%.

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<li>Herget, G. W., Neuburger, M., Plagwitz, R., Adler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+content%2C+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">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/pubmed?term=((Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts%3A+postmortem+examinations+of+human+myocardium+in+old+age%5BTitle%5D)+AND+%22Mol.+Cell+Cardiol%22%5BJournal%5D)">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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature. 29, 1011-1018 (2011).</li>
<li>Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7, 61-66 (2010).</li>
<li>Fransioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+of+the+c-kit-Positive+Cell+Response+to+Pathological+Challenge+in+the+Myocardium%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium.</a> Stem Cells. 26, 1315-1324 (2008).</li>
<li>Elliott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NKX2-5%28eGFP%2Fw%29+hESCs+for+isolation+of+human+cardiac+progenitors+and+cardiomyocytes%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.</a> Nature Methods. 8, 1037-1040 (2011).</li>
<li>Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+Cardiomyocyte+Repopulation+by+Extracardiac+Progenitors+in+Transplanted+Human+Hearts%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts.</a> Circulation Research. 90, 634-640 (2002).</li>
<li>Srsen, V., Fant, X., Heald, R., Rabouille, C., Merdes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Centrosome+proteins+form+an+insoluble+perinuclear+matrix+during+muscle+cell+differentiation%5BTitle%5D)+AND+%22BMC+cell+biology%22%5BJournal%5D)">Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.</a> BMC cell biology. 10, 28(2009).</li>
<li>Spoelgen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+flow+cytometry-based+technique+to+measure+adult+neurogenesis+in+the+brain%5BTitle%5D)+AND+%22Journal+of+neurochemistry%22%5BJournal%5D)">A novel flow cytometry-based technique to measure adult neurogenesis in the brain.</a> Journal of neurochemistry. 119, 165-175 (2011).</li>
<li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development%5BTitle%5D)+AND+%22The+American+journal+of+physiology%22%5BJournal%5D)">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> The American journal of physiology. 271, H2183-H2189 (1996).</li>
<li>Olivetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aging%2C+cardiac+hypertrophy+and+ischemic+cardiomyopathy+do+not+affect+the+proportion+of+mononucleated+and+multinucleated+myocytes+in+the+human+heart%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart.</a> J Mol Cell Cardiol. 28, 1463-1477 (1996).</li>
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No conflicts of interest declared.

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><tbody><tr><td>&lt;strong&gt;1. Lysis Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name of the reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.32 M sucrose</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-HCl (pH = 8)</td><td></td><td></td><td></td></tr><tr><td>5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>5 mM magnesium acetate</td><td></td><td></td><td></td></tr><tr><td>2.0 mM EDTA</td><td></td><td></td><td></td></tr><tr><td>0.5 mM EGTA</td><td></td><td></td><td></td></tr><tr><td>1 mM DTT</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;2. Sucrose buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name of the reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2.1 M sucrose</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-HCl (pH = 8)</td><td></td><td></td><td></td></tr><tr><td>5 mM magnesium acetate</td><td></td><td></td><td></td></tr><tr><td>1 mM DTT</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;3. Nuclei storage buffer (NSB plus)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name of the reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.44 M sucrose</td><td></td><td></td><td></td></tr><tr><td>10 mM Tris-HCl (pH = 7.2)</td><td></td><td></td><td></td></tr><tr><td>70 mM KCl</td><td></td><td></td><td></td></tr><tr><td>10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>1.5 mM spermine</td><td></td><td></td><td></td></tr><tr><td>Isotype rabbit IgG- ChIP Grade, #ab37415</td><td>Abcam</td><td></td><td></td></tr><tr><td>Rabbit anti-PCM-1 antibody, #HPA023374</td><td>Atlas Antibodies</td><td></td><td></td></tr><tr><td>Donkey sec. antibody, anti-rabbit Alexa 488 Fluor, #A-21206 or equivalent sec. fluorescent antibody</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td></td><td></td></tr><tr><td>cell strainers 30 &amp;mu;m, 70 &amp;mu;m and 100 &amp;mu;m</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Glass douncer (40 ml) and pestle &quot;L&quot;</td><td>VWR (Wheaton Industries Inc.)</td><td></td><td></td></tr><tr><td>T-25 Ultra-Turrax Homogenizer</td><td>IKA Germany</td><td></td><td></td></tr><tr><td>Dispersing tool S25 N-18 G</td><td>IKA Germany</td><td></td><td></td></tr><tr><td>Beckman Avanti Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Falcon Tubes 15 ml and 50 ml</td><td>VWR</td><td></td><td></td></tr><tr><td>Beckman Centrifuge Tubes #363664</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>JS13.1 free swinging rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Influx cytometer</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Tube Rotator</td><td>VWR</td><td></td><td></td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

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.

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 ...

The overall goal of this procedure is to isolate cardiomyocyte nuclei from postmortem tissue. This is accomplished by first homogenizing the tissue with a probe homogenizer and a glass downer, followed by several filtration steps. The second step is to isolate the nuclear fraction from the other cellular fractions by density sedimentation centrifugation.Next cardiomyocyte nuclei are immuno labeled with antibody derived against PCM one. The final step is to isolate the labeled cardiomyocyte nuclei by fluorescence activated cell sorting. Ultimately just prior to further downstream manipulation, the purity of the cardiomyocyte nuclei is confirmed by re-analysis of the sorted nuclear fractions.The main advantage of this technique is that cardiomyocyte nucle can be analyzed equally well from fresh or frozen archive tissue in a high throughput fashion. DNA based studies in cardiomyocytes are especially challenging for several reasons. Firstly, cardiomyocytes do only comprise about 20 30%of the cells in the heart.Moreover, they are to a significant degree nucleated. Moreover, cardiomyocytes are to a significant degree polyploid. This method described here do overcome all these hurdles.Before beginning the procedure. Coat ultracentrifuge tubes with 10 milliliters of a 1%BSA plus PBS coating solution. After rotating the tubes for 30 minutes, remove the coating solution and let them air dry.Begin the cardiac nuclei isolation procedure by using a scalpel to dissect the left ventricle from a snap frozen mouse heart Trim the dissected specimen into small segments, and then transfer the tissue pieces into a 50 milliliter conical tube filled with 15 milliliters of lysis buffer. Next, homogenize the heart tissue with a probe homogenizer at 24, 000 RPM for 10 seconds, and then dilute the homogenate with an equal volume of lysis buffer up to 30 milliliters. Transfer the homogenate to a glass down homogenizer using eight strokes from a large clearance pestle to free the nuclei.Then pass the crude nuclei. Isolate first through a 100 micron cell strainer, then through a 70 micron cell strainer into a fresh 50 milliliter conical tube. Now spin down the crude nuclei isolate for 10 minutes at 700 times G and four degrees Celsius.Invert the tube to carefully remove the supernatant and then wipe the inside of the tube with a paper towel. Being careful not to disturb the nuclei pellet. Pipette the solution up and down several times to resuspend the crude nuclei.Isolate in five milliliters of sucrose buffer, and then add a further 25 milliliters of sucrose buffer to the nuclei suspension. Next, add 10 milliliters of freshly prepared sucrose buffer to one of the previously prepared coated ultracentrifuge tubes. Then carefully overlay the buffer with the resuspended nuclei pellet solution.After balancing the tube in a free swinging rotor, place the rotor into a high speed centrifuge and spin the nuclei sample for 60 minutes at 13, 000 times g and four degrees Celsius. When the spin is complete, carefully remove the tubes from the rotor and again, invert them. To discard the supernatant, wipe the remaining debris from the inside of the tube with a paper towel as before, and then resuspend the nuclei pellet in one milliliter of nuclei storage buffer.Before beginning the immunos staining procedure, transfer a 20 microliter aliquot of the nuclei sample to a fax tube. Add 980 microliters of nuclei storage buffer to the aliquot and label the tube negative control. Now add anti PCM one antibody to the nuclei sample and the negative control tubes at a dilution of one to 500.Then incubate both tubes at four degrees Celsius overnight. The next day. After washing the tubes with the nuclei storage buffer and discarding the supernatant resus, suspend the nuclei pellets in another milliliter of nuclei storage buffer.Then add anti rabid fluorescent secondary antibody to the cell samples at a one to 100 dilution and incubate them at four degrees Celsius for one hour. After washing the tubes with nuclei storage buffer, resus suspend the pellets in one milliliter of nuclei storage buffer for flow cytometric sorting and analysis after coating nuclei, collection tubes with BSA and PBS as at the beginning of the video begin by filtering the sample and the negative control through individual 30 micron cell strainers. Just prior to loading, add A DNA stain.Add a one to 500 dilution for identification of the nuclei populations. Load the negative control onto the flow cytometer. Use the forward and side scatter parameters to place the first gait for defining the cardiac nuclei similar to the DOT plot shown here.Then use the forward scatter and forward scatter pulse width parameters to create a second gate around the nuclei singlets, similar to the DOT plot shown here. Now load the immuno labeled sample and placed the third gate for differentiating the PCM one positive cardiomyocyte nuclei from the PCM one negative non cardiomyocyte nuclei. Use the nuclear DNA stain to analyze for DNA content and to perform cell cycle analysis similar to the analysis illustrated by this histogram.After sorting nuclei based on their PCM one positivity, place the sorted nuclei on ice and reanalyze an aliquot of each sorted sample to determine the sorting purity. Finally, spin down the sorted nuclei in their collection tubes for 15 minutes at 1500 times G and four degrees Celsius. After discarding, the supernatants resus suspend the nuclei pellets in a buffer compatible with the desired downstream application.In this first figure, an adult mouse heart was stained with a nuclear dye and antibodies against PCM one, the murn heart transcription factor N KX 2.5 and myosin heavy chain or MHC. Here a closeup of the overlay from the previous figure is shown. Note how the PCM one labeled nuclei in white express the transcription factor NK X 2.5 in red and are surrounded by cardiomyocyte cytoplasm in green.These data together demonstrate the accuracy with which PCM one staining identifies cardiomyocyte nuclei. The inset highlights the epi nuclear staining pattern of PCM one in myocyte nuclei as further indicated by the arrows in the experiment depicted in these next three figures, cardiac nuclei isolates were stained with the DNA stain DRAC five, and with antibodies against PCM one. Here, the overlay of the previous two figures, the arrows once again highlight the epi nuclear staining pattern of PCM one in the isolated cardiomyocyte nuclei.This dot plot demonstrates that approximately 30%of the nuclei isolated by this method are cardiomyocyte nuclei as indicated by their PCM one labeling. Finally, as these histograms illustrate, mouse cardiomyocyte nuclei are primarily diploid as indicated by the two N peak and only a small subset is tetraploid as indicated by the smaller four N peak. One of the advantages of this technique is that it can easily be adapted for other species.As for humans.

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JoVE Journal

Medicine

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

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.

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

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&lt;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

Coronary endarterectomy tissue samples were processed to isolate single nuclei using an optimized dissociation protocol. The integrity of the isolated nuclei was validated through three complementary approaches: trypan blue exclusion staining to assess membrane integrity, DAPI-based confocal microscopy to confirm nuclear morphology, and flow cytometry to quantify nuclear yield and purity.

Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

Learn to isolate cardiomyocyte nuclei from postmortem tissue using homogenization, density sedimentation centrifugation, and fluorescence activated cell sorting with PCM-1 immunolabeling. This method achieves high purity for downstream cardiac research.

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

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