THP-1 differentiated by PMA - (Nov/07/2013 )
I just recently work on THP-1 as well as mammalian cell culturing.
I have a problem with THP-1 as it's non-adherent cell line, after settle it down in well, I will have to induce it with PMA. The purpose is to have differentiated THP-1 cells stick evenly after incubation. But after THP-1's settling, the cells accumulate in the center of the well, and especially after PMA treatment, cells differentiate and stick to big clumps.
Anyone have experience with this please help me!
When we plate out THP-1 into wells, we always add the PMA to the stock solution of THP-1 cells to be used then pipette into the well gently. This results in a good spread of the cells.
Thank you .I will try this
But I have a question to ask about your method: if we put PMA into stock solution of THP-1, will the cells clumps even when they are not attached to the plate surface (form "flying" clumps)? And usually in culturing other cells, I see that people may have to pipet their cells to make them well distribute (or shake the plate gently), do you have to include any techniques to make them well distribute?
After placing the plate into the incubator, I would very gently move it back and forth, and then left to right to distribute the cells evenly. It is then important to close the incubator door GENTLY and put a note on it so that everyone else does it too. Slamming the door results in cell clumps in the middle of the wells.
Thank you for your advice
I tried tips that you provided.
Now, after 22hrs of PMA induction, I have just about 30% of cells stuck on the plate. other floating, about to form clouds of clumps. Any tips,please?
PMA differentiated THP-1 cells are the main cells I work with - what concentration of PMA are you using and how long are you allowing cells to differentiate? I allow 72 hours for cells to differentiate in PMA treated media, then wash and change to normal media and allow the cells to rest overnight and get about 90% adherence and differentiation. The following article may be of use:
Hope this helps.
I used to differantiate THP-1 cell several time. Firstly, how much PMA you used . I think accurate differantiation occured with 200 nM/10e6 cells. However what is the protocol you used . after 22 hrs, there will be no differantiated cells. what I did. is to leave the cells in PMA for 3 days. then wash out the medium and excess PMA and after that add new fresh medium for 2-3 days but observe every day.