Hello,
I've been trying to differentiate my THP-1 cells for days. I've seeded 2.10^4 cells/well in a 96 well plate, then I treated them with PMA concentrations ranging between 10-400 nM. I waited a whole weekend and they didin't differentiate. I tried again with higher concentrations but I saw an article that says it can influenciate the macrophage response to LPS and it becomes more sensitive to stimulation.
My THP-1 cells are being cultured in DMEM 10% SBF and they're growing well.
I appreciate all the help I can get!
Hi Jordana,
We used to differentiate our THP-1 cells by incubating them with 150 nM of PMA (P8139; Sigma) for 24 hours in RPMI medium with 10 % heat-inactivated FCS. After 24 hours, the medium with PMA was removed and fresh RPMI medium with 10 % heat-inactivated FCS was added for another 24 hours before any treatment. Have you any indication that your PMA is active as I don't see why the cells would not differentiate in DMEM ?
Best,
Damien
hi,
I worked on THP-1 for three years as a part of my Ph.D. and I used 50ng/ml of PMA to differentiate THP-1 in RPMI containing 10% FCS for over 48h and it was enough to differentiate the cells at a 100%, as you aware the cells are not adhesive by adding PMA at 50ng/ml will change the cells to adhesive one as indication for the cell differentiation.
regards
Hi Jordana,
We used to differentiate our THP-1 cells by incubating them with 150 nM of PMA (P8139; Sigma) for 24 hours in RPMI medium with 10 % heat-inactivated FCS. After 24 hours, the medium with PMA was removed and fresh RPMI medium with 10 % heat-inactivated FCS was added for another 24 hours before any treatment. Have you any indication that your PMA is active as I don't see why the cells would not differentiate in DMEM ?
Best,
Damien
Different cells utilize different inducers/pathways of differentiation. You might also consider the cAMP pathway: methyl-isobutylxanthine (IBMX). Also consider hexamethylbisacetimide (HMBA). Keep trying!
If it is distinctly a protein kinase C mediated pathway in your cells, for a unique twist, try bryostatin. It will activate/downregulate PKC-a with very different kinetics than PMA.
Thanks for your answer, @Nawzat! I've tried 50 ng/mL before and it didn't work! (apparently my PMA wasn't good)
@James, your point is very interesting. I didn't know that THP-1 cells could be differentiated by things other than PMA and Vit D. I'll see some articles about that. Thank you so much!
@Damien, you were right! I've tried two different PMAs solutions that we had and apparently they weren't active as I thought, so we bought a new one from Sigma. I tested it and it worked!! In 48-72 hours they've differentiated at 25 nM! Thank you! I'm very happy now!
Thank you all so much!
Regards,
Jordana.
Jordana, as pointed above, undifferentiated THP-1, as non-adherent cells, should be grown in RPMI medium instead of DMEM. I would try change the growth medium as well.
Good luck!
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