Hello friends,
I have been carrying out cardiomyocyte isolations for a few weeks now and I want to get a homologous mixture (or at least 90%+ purity) of just cardiomyocytes to carry out some cyclic AMP assays in response to some ligands. I have read up on FACS sorting and Percoll gradients...I was wondering if anyone can recommend the best method?
Dear Joe Davidson
if you wish to culture cells to perform cyclic AMP assays then I would recommend to go ahead with purification steps based on Percoll gradient- the process takes longer but the cell viability is much better in our experience. I prefer to use FACS when I intend to harvest the cells directly for RNA/Protein etc.
All the best!
Shambhabi Chatterjee Thanks so much for the reply.
I was curious do you have a protocol for the Percoll gradient and the FACS sorting?
Thanks so much for your wisdom.
Dear Joe Davidson
we use the Miltenyi dissociation kit (https://www.miltenyibiotec.com/DE-en/products/neonatal-heart-dissociation-kit-mouse-and-rat.html#130-098-373 ) for cardiomyocyte isolation according to the manufacturer's protocol and combine it with percoll gradient to increase purity of CMs .
Pls have a look at the section "Percoll gradient separation" in this protocol- http://hivebench-star-protocols.s3.amazonaws.com/protocols/1171.pdf
for FACS- after the miltenyi isolation steps instead of seeding the cells we proceed with cTNT antibody staining and go ahead with FACS sorting.
Both the conjugated antibody (https://www.miltenyibiotec.com/DE-en/products/cardiac-troponin-t-antibody-anti-human-mouse-rat-reafinity-rea400.html#conjugate=pe:size=30-tests-in-60-ul ) or monoclonal cTNT antibody (https://www.abcam.com/cardiac-troponin-t-antibody-1c11-ab8295.html) has worked well for staining rat CMs.
If you need live cells then you can't fix them and in that case CORIN antibody (Article Isolation and characterization of hESC-derived heart field-s...
) might be a better choiceHope this helps.
Some flow cytometers have the ability to count cells. How are you analyzing your cells?
Sorting will get you better purity than a density gradient such as Ficoll or Percoll. The density gradient method is cheaper and doesn't require special equipment - but if your application needs >= 90% purity then consider a sorter.
Both isolation methods have an impact on cell health. If you are sorting with a droplet sorter (like the Sony or BD) the cell viability can be an issue plus the significant time spent cleaning and setting up the instrument. Tyto cell sorting is mechanical, keeps your cells cold and in a buffer of your choice.
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