21 replies to this topic

[XD88]

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!!

[bob1]

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

[SuMi]

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!

[XD88]

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

[SuMi]

I get mine from Sigma

[XD88]

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

I get mine from Sigma

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

[SuMi]

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

[suncherry]

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.

[SuMi]

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.

[XD88]

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

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.

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...

[suncherry]

I'm doing immunofluroscent on THP-1 macrophage but the autofluorescence is too stronge to see the target staining. Is autofluorescence common in THP-1? What's the reason? How to dimish it? Thanks.

[bob1]

Autoflourescence isn't usually a property of the cells, often it is caused by residual formaldehyde from fixation - wash your cells more after fixing.

[farhanah]

Hey!

I have several problems activating my THP-1 cells with pma!

-my cell viability is really poor,probably due to the scrapping.And ppl say that we shouldnt use trypsin,so what else can we use to lift the cells up from the surface?

-i am using 12ng/ml.Is that too little? Because after facs,my cell surface markers are not coming up.

-what markers do u guys suggest i use? For now,im using HLA-dr,CD15 and CD16. And they dont seem to appear after activation of THP-1.

[SuMi]

I use a concentration of 50ng/ml PMA. Media is changed after 24 hours and then cells are cultured for a further 48 hours. However, there was a paper that showed that 5ng/ml was sufficent to induce differentiation.

To detach the cells, I remove the supernatant and add 5mM EDTA in PBS to the cells. I then incubate the cells for 15 to 20 mins at 37oC and the cells just pop off! I remove the cells and then wash each well with PBS + 10% FCS as the EDTA makes the cells sticky and this removes the remaining cells as well.

As for markers, CD14 is upregulated in differentiated THP-1s.  I used macrophage mannose receptor (CD206) when I was culturing human macrophages but I'm not sure if THP-1s express this.

Hope this helps.

Edited by SuMi, 23 September 2009 - 07:31 AM.

[beth]

Hi,

You need to keep the THPs in PMA for 72 hours. If you change the media, add more PMA. I add 20ng/ml PMA (Sigma) to my cells. I check on them 24hours later. If still quite a few floating, I just add another 20ng/ml PMA without changing media to get these to stick down. Then the next day, I usually change the media and add more PMA. I also keep PMA in my cultures when doing treatments. I have no problem with them. They become nice and macrophage like. At 24 hours they should mostly be stuck down and as time progresses they will flatten out and be less bright and have projections.