I am new working with THP-1 cells. I explain a little bite with I do with them. First, I differentiate the THP-1 to M0 with PMA, once they are differentiated, I detach them by scrapping the petri dish. Then I mix the M0 cells with myofibroblast in order to have a co-culture. Finally, I embed them in a hydrogel and let them for 3 weeks.
My problems are:
1. Cell proliferantion after been differentiated
- I thought that after THP-1 cells have been differentiated to M0 with PMA cells stop proliferation. However, when they are embedded in the hydrogel I have seen that M0 cells start to from aggregates inside the hydrogels and there are a lot of suspended cells in the medium. This means that can M0 cells divide? Could it be because THP-1 cells are not properly differentiated to M0? Or because after I while without any PMA, do M0 cells go back to THP-1 cells?
2. I have problems with the CD68 antibody to label specifically the M0 cells.
- In order to know the cell distribution in the hydrogel I have done an Immunofluorescence. To do that, I have used the CD68 antibody to label specifically for THP-1 cells and collagen type IV to label myofibroblast. However, after I have done the immunofluorescence, I have found that myofibroblast express CD68 cell marker. So, the CD68 antibody labels both types of cells and it isn’t useful for me because I cannot differentiate them. Do you know a cell marker that is expressed only in the THP-1 (M0) cells? Could you suggest an antibody for doing immunofluorescence that works?
It would be very useful if you could give me some advices.
Thank you in advance!
Anna
Hello Anna,
what you are doing sounds very interesting and challenging.
For your first question, you mentioned one possibility. Have you considered that your M0 cells translocate to specific sites to form clusters? If you have a system that is differentiating at the moment, you can follow up a selected region over a few days, to confirm what is happening.
For your second question just check the link
https://www.uniprot.org/uniprot/P34810
Unfortunately, I cannot advise other antibodies since this is not my field of expertise, but I am sure there are several other commercial markers.
Good luck with your experiment.
Kind regards
Olga
If you haven't already seen them, this earlier RG thread:
https://www.researchgate.net/post/Which_are_the_best_differentiation_markers_in_Thp-1_and_U937_cells
and this article:
https://www.bdbiosciences.com/documents/BD_Multicolor_MonocyteMacrophageDiff_AppNote.pdf
might be helpful in identifying a unique marker. There are then many possible sources for antibodies against the marker(s).
To your first question:
1. What is the PMA protocol you are using?
2. It may be worth checking THP-1 derived macrophage viability in the hydrogel. I have experienced THP-1 clumping upon cell death.