Hi,
I am trying to use a flow cytometer with primary cardiac myocytes.
I'm struggling to get a defined cluster that I can be sure represents the live cells on the FSC-SSC plot. All I get it is a dense cluster at the lower end of the FSC / SSC scale (which I assume are the dead cells?) then noise.
Has any one got any basic advice on this (I am completely new to flow cytometry!). Is there something special I need to be doing when using cardiomycoytes (compared to say red blood cells)?
Best
Dave
Hi David
I've never worked with cardiomyocytes but the two main things you should do is:
- Isolate single cells on your analysis. You should have a FSC H vs FSC A histogram in order to gate only singlets. I assume you are using cells isolated from tissue and not a cell culture line. The tissue dissotiation protocol can lead to cell clumping so it's import to be sure you have a single cell suspension.
- Use a viability dye like 7AAD or Propidium Iodide to assure you are analyzing only live cells. Again the tissue digestion protocol can cause cell death and it's import to eliminate those cells during your analyzes.
In conclusion, first gate on single cells, than gate on live cells and then gate on your specific antibody (fluorochrome).
Yes I agree - 7AAD for viability and put your dead cells in a dump channel.
Hi David, Have you managed to sort cardiomyocytes using FACS in the end, I am also interested in trying to do this so would be great to get some advice. Many thanks.
Davor Pavlovic Dear Davor, did you sort out cardiomyocytes with FACS ? as I'm looking for surface markers to do so !
we have not yet. If we get anywhere with this I will send information.
Besra Samy
If you are working with adult cardiomyocytes then it will be difficult to analyze/sort them due to their size. They range in size from approx 70um to 200um. If they are adult cardiomyocytes then it is unlikely that they will pass through the sample line or through a nozzle. What you are seeing in your facs plot is just the small debris that is in your sample. For adult cardiomyocytes you need a Large Particle Sorter e.g. BioSorter from Union Biometrica. Otherwise you could try filtering out the debris and small cells with a 40um filter and use the microscope to analyze. If you are fixing your cells you can stain with Troponin.
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