Hi,
I'm trying to make macrophages from THP-1 cells by treating PMA in culture media. This time is third time, however, the cells were not adhere to the bottom of culture plate. I seeded 1 * 10^6 cells in each wells with 2 ml of complete (10% 1x Ab in RPMI) media containing 200 nM PMA. After seeding the cells, THP-1 cells were cultured during 3 days without any treatment. and, I observed cell state in every day with microscope, however, the amount of adherent cells were less than 60%.
at the first trial of differentiation, cells were adhered more than 80%, but in nowdays, adherence efficiency is lower than previous trial.
what is the problem? the concentration of PMA? or the state of PMA? (break down?) If someone know the answer, please help me.
Hi Jun-Pyo Choi,
I have been working with THP-1 and there are many factors which can contribute to differentiation/ culture issues. I will list some factors which I believe to be critical and that you might check in your protocol:
- Have you checked if culture is clear of infection? Mycoplasma contamination can interfere with the process, you could run mycoplasma test in your culture.
- Which cell passage do you use since thawing? For our experiments, we tend to use THP-1 from passages 3 - 10 interval (P3 to P10), we have observed that lower or higher passages impact on assay reproductibility.
- We do 2 weekly passages of THP-1 and the cells are always kept within the range of 1x10e5 to 1x10e6 cells/mL. In fact, under/over concentration of culture might impact in the differentiation/cell viability.
- I believe the PMA concentration you use is enough, actually, in our experiments we use 1/4 of this concentration (50nM) for 3 days differentiation and the cells adhere and differentiate; so perhaps you could try lowering the PMA concentration for a test.
I hope this could help you somehow.
Cheers
Dear Caio H Franco,
Thank you for your specific and kind response. There was no bacteria contamination. So, exclude this issue, there is three issued should I check.
Actually, THP-1 cells that I used were obtained from Lab. co-worker, so I couldn't count the exact passage. If regarding that the first cell I received from co-worker is the passage 1, all of cells were not exceed the passage 10 and in the range that you mentioned.
And, I fixed the cell number 1*10e6 in each well, and increased the volume to 2~3 mL. when exceeding the 2.5 mL, the differentiation efficiency was good. However, 1.5 day after stimulation with PMA, floating cells were appeared. So, as your recommendation, I'll try lowering PMA concentration for next test.
Thanks.
Hi Jun-Pyo Choi
1. Have you seen cell death or apoptosis in the culture?
2. Actually, PMA can be toxic to cells.
3. So, you must try lower concentration and tray at least 2-3 concentraion then you will be come out with the right concentration for your lab condition.
Thank you,
Good luck
Hello,
100-200 nM PMA is considered a moderate dose, sufficient to activate undifferentiated THP-1 cells within a time frame of 30 min. One of the best known effects of PMA on THP-1 and monocytes is ROS induction.
Article HKOCl-3: A fluorescent hypochlorous acid probe for live-cell...
With a moderate dose of PMA, at least partial (therefore premature) activation is expected. Therefore, it is also not surprising that some cells are either out of replication (cell cycle) or out of differentiation (attachment, etc.), but into a weak inflammatory phenotype followed by cell death.
Optimized THP-1 differentiation has been discussed many times in this RG forum and beyond.
One recommended protocol involved a very low dose of 5 ng/mL PMA, for either 3 days (then wash off) or 5 days (no wash). You may take a look at:
https://www.researchgate.net/post/Once_a_THP-1_cell_is_differentiated_to_macrophages_by_PMA_how_long_will_they_stay_as_macrophages
Also, make sure that your seeding density is not too high (you can try out 1-5 x 10^5 cells/mL). 1x10^6 cells/mL is a saturation density for THP-1 (considered a cap density by ATCC, beyond which growth is suboptimal). When the cells get differentiated, their sizes increase at least 4-5 times or larger, and cells spread out on the dish surface. If at seeding the cells are already crowded (observe under a microscope), then you cannot expect the differentiated cells to be content.
Good luck
@ Deepak K. Goyal
Thank you for your comment!
I also have considered that possibility and tried more than 2 doses. As a result, I have founded optimal dose, 100 - 150 nM.
Nai-Kei Wong
Thank you for your very specific and helpful advice!
Cause of your advice, I can solve the fundamental problem in my culture!
Dear Jun-Pyo Choi ,
I hope this article will be helpful.
I have tried with this protocol and i got good results.
All the best.
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