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.
