Have been trying to induce THP-1 using PMA at different concentrations (range from 5 to 50 ng/mL) but after 48h all the conditions except the control are mostly dead, checked by trypan blue and flow cytometry.
Media: RPMI 1640 + FBS + P/S + 0.05mM mercaptoethanol
0.5x10^6 cells / mL of media in a 6-well plate.
Any help? From what I read on the literature these cells should not be these hard to stimulate with PMA.
Thanks in advance
Hi Eduardo,
Sorry to hearing that. Did you try stimulating the same condition by different agonist such as LPS, IL-4 or IL-6? to make sure the issue is due to the stimulation or may be the cells?
No, I have not yet. That is indeed a plan going forward with those cytokines
Good Luck. Please share the findings with me if that possible. It's worth to my experience.
Long time ago, I used THP-1. THP-1 response and viability vary with PMA conc. Maybe, you are adding too much PMA.
I found, 2-ME is not really needed.
I have already induced THP-1 with PMA using 25ng/ml at a density of 200 thousand cells/ml. I used RPMI medium supplemented as specified by ATCC, and using DMEM-F12 medium also worked. It was 48 hours with PMA, followed by 24 hours with PMA-free medium.
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