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

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.

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

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.

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.

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.

suncherry on Jun 30 2009, 08:48 AM said:

Uh, THP-1 cells are a very stable cell line. Passage number does not matter. It has been around since the 1980's. Do you really think that you are getting passage 1 wherever you get them from? Passage number is more an issue for primary cell culture or self-immortalised cells. Just make sure you always keep stocks which are not treated with anything to bring up fresh cells when needed. I have never ever noticed anything change with passage of these cells and must have passaged them hundreds of times. I culture mine in RPMI + 10% FBS and pen/strep. Obviously, over time if you culture cells under certain conditions you may get changes, which is why results can vary from lab to lab, due to different FBS, etc.

http://en.wikipedia.org/wiki/THP1_cell_line

beth on Feb 5 2010, 07:43 AM said:

I am trying to standardize thp 1 differentiation using PMA. Can anybody tell me should I use treated or non treated plates for the treatment? and also what are the other CD markers which can be used to monitor this differentiation other than CD 14.

Hi SuMi!

I am trying to differentiate THP-1 into macrophages using PMA.

I get the adderent cells after 72h of incubation.
After that time, I removed the supernantant and I also used PBS+EDTA 5mM to detach the cells. However, I just detach cells incubated with 10nM of PMA, I didn't get "free" macrophages from (20, 40, 80 and 160nM of PMA).
I also used trypsin, and I get some detached cells, however, a lot of them continued adherent in the plate.

What do you advise??
Did I use higher concentrations of pMA, or higher incubation time?

Many thanks in advance.

Best regards

samasya on Tue Jul 13 13:41:32 2010 said:

Macrophages derived from PMA treated THP-1 monocytes are very adhesive and they sticked to any surface once activated, any culture T75/T25 flask/well surface are good enough for these macrophages, so there's no need to pruchase extra collagen/matrix treated plate for them. Apart from CD14, you can also try common macrophage marker CD68 ...from what i did before, THP-1 cells and PMA activated form are distinct in terms of morphology , protein and cytokine expressions .......no problems in distinguishing them at all

I treat my THP-1 with PMA. I prepared 50 ml medium RPMI with 10% inactivated serum, antibiotics solution and 50 ng/ml PMA. I would like to know If I can store this medium in refrigerator for a few days? MAybe I should do a fresh medium for every experiment….Please help

Anula