I have been having issues with my THP-1 cells for most of this year - when I try to differentiate them with PMA into macrophage-like cells about 1/3 of the cells do not adhere properly (either floating or loosely attached) and most of these seem to die. I have tried to wash these cells off and use the ones that are adhered down but during experiments these are not happy and will round up and de-attach.
I have tried everything I can think of - gotten new PMA, tried different PMA concentrations, all new media and FBS, tried different cell densities, different culture plates etc. but nothing seems to make a difference. The cells are also negative for mycoplasma. I have experienced this issue with different batches of THP-1s, including a recently ordered batch from ATCC, which suggests it could be the way I am culturing the cells but I have not changed anything from the way I normally culture them - and they have been fine for 2 years prior to this issue.
I culture the cells in RPMI + L-glut + 10% FBS. I typically use 50 ng/ml PMA for 3 days and then an overnight rest before experiments (but I see this issue within 24hrs of PMA addition). My monocyte cultures do not exceed 1x10^6 cells/ml prior to seeding.
Has anyone come across this issue before?
Thanks in advance for any help with this.
Hi Richard Allen
I briefly wanted to reach out concerning your issues. Please consider the following:
1. THP-1 cells are really susceptible to freeze thaw damage and even if frozen and stored correctly seem to have diminished viability post 6 months storage.
2. THP-1s are happiest between 1x105 cells/ml to 1x106 cells/ml (2x106 cells/ml maximum, they do make some kind of autocrine acting growth factor so lower seeding will impair growth and use beyond 5 passages does not yield ideal results either.
3. The ideal media according to ATCC is RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate and supplemented with 0.05 mM beta-mercaptoethanol and 10% FBS.
4. For differentiation with PMA consider a cell concentration of 2x105 cells per ml, use PMA at a concentration of 100 nM and allow to differentiate for 3 days.
For more details consider the following:
http://www.bowdish.ca/lab/protocols/cell-tissue-culture/
I always had great results following it.
All the best & good luck with your experiment.
Cheers,
Michael
Hi Michael Förster , thanks for the response.
1. These cells were recently ordered from ATCC and have only been recovered from freezing once, so I would be surprised if this is the cause of the issues. We have used THP-1s that have been frozen for over a year with no issues before in our lab.
2. I keep my cells between 4x10^5 and 1x10^6 cells/ml usually and they never exceed 1.5x10^6 cells/ml so I don't think cell density is the issue either.
3. I am currently in the process of trying the ATCC modification of RPMI but we have never had issues using RPMI + L-glut + 10% FBS in the lab before so again am skeptical this is the issue.
4. For most of my experiments I do indeed seed at 2.5x10^5 cells/ml with a concentration of 50 or 100 ng/ml PMA - but I have tried varying the concentration of PMA as well but see no difference in viability within 24hrs.
Thank you for the advice, and any further insight would be greatly appreciated!
Richard
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