I am working with THP-1 cells at the moment and am having trouble with maintaining a high confluence once I seed them on a 24-well plate. I usually try to seed around 100,000 cells per well and leave them overnight to adhere. However, I have been finding that they do not adhere very strongly. As a result, whenever I have to switch the media and wash the cells, I am left with a very low confluence. I have been very gentle with the wash and while aspirating the media, but I keep on running into this problem. Does anyone have any suggestions to try and fix it?
Hi Anna,
since THP-1 cells are monocytes they are non-adherent (if not activated/differentiated to macrophages). You need to keep them in suspension as described https://www.lgcstandards-atcc.org/products/all/TIB-202.aspx#culturemethod. You do not need the mercaptoethanol but might want to add amino acids.
In our hands it always worked well to keep 50% conditioned ("old") media and add 50% fresh RPMI. However, you need to transfer them into a collection tube and centrifuge gently to collect the cells and replace media.
Boris
Hi Boris,
Thank you for the link you shared with me! I have been adding the cells to the 24 well-plate to differentiate them to M1 and M2 macrophages. Would you recommend differentiating them first, and then seeding them (as they should be adherent by then)?
Thank you again!
Hey Anna,
depends on what you are up to. If the different wells are supposed to be replicates of one experiments it might be good to treat the complete batch for differentation and then seed. You might loose some cells though. There is one older thread on the topic here "Why do my monocyte-derived THP-1 macrophages die so much?". Maybe have a look.
Good luck
Boris
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