Differentiating Thp-1 into macrophage - (Jun/24/2009 )

I've tried induce differentiation of Thp-1 into macrophage by culturing the cells with 100 nM PMA for 24 hours then replacing the PMA media with regular RPMI Thp-1 growth media to keep the attached cells growing/differentiating. I noticed some adherence after a day of PMA stimulation but the cells started detaching after media change. Does anybody have any suggestions? Please help!!

Macrophages are motile aren't they? Perhaps try growing them as a suspension cell line if they are motile.

When I convert my THP-1s into macrophages I stimulate with PMA for 72 hours. I add 50ng/ml PMA for 24 hours, change the medium to regular complete medium and then leave them for a further 48 hours.
It's pretty hard to get them to detach after that!

Thanks guys! May I ask, where do you get your PMA from?

I get mine from Sigma

SuMi on Jun 25 2009, 06:13 AM said:

Can you tell me your serum content during differentiation and also your starting cell density? Thanks!!

I use 10% serum throughout and I usually seed 1e6 cells/ml with 3ml/well of a 6-well plate.

Hi,
I met the similar problem that after change the medium it detached. I have induced THP-1 into macrophage for several months. Induction methods is the same as above. Only recently the problems occured. Do you think it is because the cell itself? Because the cells have cultured and passaged for more than three months while induction methods doesn't change.THP-1 cell can passage endless? Please help.Thanks a lot.

I'd say its definitely the passage number. I only ever use my cells for 10-12 weeks as I had problems with autofluorescence from the THP-1s when they were passaged for too long. If I were you I would start from new stocks and I bet they will differentiate fine. Hope this helps.

SuMi on Jun 30 2009, 12:49 PM said:

I've only been using my cells for less than 8 weeks though... have you tried using fewer cells/ml? I'm thinking it might be the cell density...