In my first experience, I cultured the PBMCs and isolate the adherent cells to the flask after 10 hours. After about 2 days the morphology of cells changed from a round small shaped-cells to the cells which you can see in the attached figures. Also, I treated these cells with GMCSF in 5ng/ml concentraion, but I don't sense any change in the cells. Does anybody have any comment for me?
Hi Majid I'm not entirely sure as to the identity of these cells.
However I doubt that you will get a pure monocyte population using this approach as this technique is often used to isolate Mesenchymal Stromal Cells (MSC) which have a fibroblast morphology (so they don't look like your images). You will also get Macrophages too this way.
Are the cells you observe proliferating? IF so are they forming colonies?
A lot of these look almost like some kind of "debris" rather than cells. Have you stained any with a nuclear dye to see if they contain a nucleus (and determine the nuclear morphology)?
Dear Gary
Thank you for your valuable comments. I don't find any proliferation in these cells after about 10 days, even in the presence of GM-CSF. I think that the GM-CSF just help these cells to survive in the culture. I try to use a nuclear dye and evaluate their nuclear morphology. But do you have a better protocol to culture these cells?
Hi Majid - sorry for delay in replying to you (some problem with my computer).
Most monocytes are isolated via antibodies although I did find this article
"A Simple Method for Human Peripheral Blood Monocyte Isolation "
which appears to use Ficoll only:
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762000000200014
I hope this helps!
Gary
Hi Gary
Thank you for your comment. It's very helpful. Also, I found the below link:
https://www.researchgate.net/post/Which_conditions_do_I_need_to_differentiate_to_THP-1_cell_to_macrophages
All the best
Majid
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