Hi ! I wanted to do flow cytometry on primary adipocytes and hepatocytes but I cannot find any publication with protocols. Is it possible to do on primary cells ?
Hi Fiona,
Yes it is possible to do flow on primary cells. I do it on stem cells as wells as differentiated cells all the time. The protocol for staining is no different than any other cells/cell line. Just do any surface or intracellular staining as you normally would. Just make sure you have unstained cells to adjust the voltages when you run the flow.
Good Luck!
Hi Fiona,
It is possible to do flow with these cells, but keep in mind that primary hepatocytes tend to be very autofluorescent in the FITC channel. Therefore I would advise you not to use a FITC- or A488-labelled antibody in your panels.
Good luck!
Anouk
Hepatocytes yes (depending on conditions) adipocytes not likely however. The other tips suggested are good for most primary cell populations, but hepatocytes and adipocytes considerably complicate flow cytometry.
Liver and adipose tissue tend to contain a lot of highly autofluorescent cells including the cell populations you are interested in but also tissue resident macrophage populations.
The size is another major limitation. Adipocytes, in particular are actually usually larger than the filters used on many modern flow cytometers. Figure out if the filter used is 100um, if so most of your adipocytes would just explode on contact. A similar thing can happen to hepatocytes during steatosis (high-fat diet feeding or genetic).
In addition, adipocytes are incredibly difficult to isolate and work with as you would another cell population, for instance lymphocytes, as they tend to start exploding in the tube during normal preps.
Overall I haven't really seen convincing adipocyte flow data. You can get preadipocytes, but the mature adipocyte is just too hard to work with generally
Thank you for all your interesting answers! I suspected that flow cytometry on adipocytes would be difficult but I did not know for hepatocytes and for autofluorescence of primary cells. Anyways, it seems that this is not an usual experiment to perform !
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