I am having problem in differentiation of THP-1 monocytes into Macrophages. THP-1 cells are growing fine. I plate them into, 6 well plate at a concentration of 1X10^6 /ml. The final PMA concentration is 100ng/ml (~ 160nM), and then I incubate them for 24 hrs. After, 24 hrs the cells tend to form clusters and they are not firmly attached to the plate. After this 24hrs, I try to remove the media (to add fresh media without PMA) and give it a wash with warm PBS, most of the cells are washed away. Please help. Thanks.
Hi Raghav,
I grow THP-1 cells this way:
- Final cell concentration 3.5x10^5 (maybe yours is too high, cells may not be comfortable at such high concentrations and attack each other)
- Differentiation with 200 nM PMA during 96 hours. During this time, I do not move them at any time (maybe your PMA incubation time is too short, cells don't differentiate well to macrophages and thus they don't attach correctly to the plate)
- Sometimes the attachment to the plate depends on the plastic used to manufacture it. Try with plates of other commercial house (we use 24-well Labclinics-NUNC plates and they work perfectly)
- Be sure that you are working with a THP-1 cell line that is fully working, without too many cell passages (THP-1 cells work correctly up to 25-30 passages)
I hope this is helpful!
I agree with Alvaro; however, you may also want to try butyric acid or vitamin D3 at 100 nM in each case. It Works well it all depends of you want to use the cells for.
Hi Raghav,
I have encountered the aggregation issue as well. Were you able to solve it? In my case I see the aggregation in treated and untreated cells. Therefore I think it is more of an issue with the plastic surface rather than PMA treatment.
Also, the cells form clusters within a few hours of plating. I have tried resuspending them every few hours. However, they still aggregate once again along the center of the well in the form a elongated strip.
Some people have suggested using poly-L-lysine coated plates. I am about to give that a try.
Let me know if you find any other solution!
Goodluck!
Hi Raghav,
I had the same problem and I am using Cell Tak, It works very well!
Good Luck!
I use bacteriological (non-treated), 60 mm dishes, 2 million cells in 4 ml medium. PMA 100 ng/ml for 3 days. Aggregation is not an issue. After 24 h cells are not yet attached.