You will observe a severe phenotypic change i.e. spreading of cells / adherence to TC dish surface as opposed to sphere shaped / non-adherent THP-1 cells; if you need to additionally confirm / estimate % differentiated cells you can fix and stain with e.g. anti-CD4 at 1, 2, 6, 24 & 48 hrs post-PMA, you should see rapid (and hopefully complete) disappearance of CD4 positivity.  Good luck!

What are you going to do with THP-derived macrophages? Usually after 72 hours after PMA stimulation all cells have macrophage signatures. In my experiments I usually use them 5 days after PMA stimulation.

Hi Vladimir, I observed the same phenotypic change i.e. 100% adherence and even their expansion. CD4 staining is a good option and i will definitely go for it. Can you please post the link of article where this phenomenon has been proved.

Thanks Mikhail, I use these cell for Leishmania infection.  

Regards,

Of course, Jiten - here is a pubmed entry that I suggest you read not just for referencing but also to learn some basic THP-1/PMA things: http://www.ncbi.nlm.nih.gov/pubmed/1999239; if you get trouble getting the paper via pubmed just google it and it will pop up in one place or another

All the best, V

Which conditions do I need to differentiate to THP-1 cell to macrophages?

Thanks Vladimir, got the article .

Hi Handon, 

there are different conditions are mentioned in literature. Most commonly used one is to plate cells with PMA (100ng/ml) for 24 hrs. After 24hr rest them in PMA free media. 

All the best,

Regards, 

Many thanks Jiten, actually I wonder if I need to differentiate THP-1 cells to macrophage. My problem is that they will be attached to the bottom of the plate after stimulated. I already will be induced to differentiation. In this case, is it important cell type which are being monocytes or macrophage ?

Hi Handon, 

Can you explain your problem more in brief, what is your experimental design so that i can suggest or help you out.

Just an example for Handan. We study inflammasomes activation and usually use human monocytes or THP-1 cells. However, there is a drastic difference between monocytes and macrophages in ability to activate caspase-1 - usually no or very little inflammasome activation in macrophages. To study details in inflammasome activation, we use monocytes and macrophages, THP-1 and THP-derived macrophages (TDM). Also, we have many THP with stable over-expression or knocking down of inflammasome-related molecules which have different expression patterns in monocytes and macrophages. In particular, one of pro-inflammatory molecules, PYRIN, is expressed in monocytes and THP, but not in macrophages and  disappeared in TDM. Over expressing pyrin in TDM helps us to better understand its role. There are many reasons why people use THP or TDM, or both. What is your experimental question? 

Jiten Verma,

In my study i have to differentiate THP1 monocyte to macrophage and also i have to infect the macrophage cell with Mtb. i am here to get your help regarding the following problems...

1. You suggested here to use PMA (100nM) for the differentiation but i am worried about the cell number. (would you please give the reference) 

2. In case of infection, the MOI is important. so is there any chance to increase or decrease the macrophage cell number during 1d incubation without PMA as you have mentioned?

Hi Hafij,

I used to seed approx 0.5 X 106  cells/per well in a 6 well plate. I always get a good differentiation with this numbers. 

For 24 well plate seed approx 0.1 X 106/well in 1 ml of medium.

Its advisable to differentiate them for 24 hrs in the presence of PMA and rest them for the next 24 hrs without PMA.

With these number of cell, i always get good infection and similar results every time.

Best of luck....

Thank you so much for your feedback Jiten.

Hi, Iam also performing some studies with THP-1 wherein I plate them at a density of 5*10^5/well in 1 ml of medium in 24 well plate. I differentiate it with PMA 25 ng/ml for 24 h and next 24 h without PMA. While performing infection studies with bacteria, while giving wahses, I observe cell loss. Does this happen with you, Iam asking since you are using an even lower cell density and hence the cells would be even more distributed?

Hi,

I always differentiate THP-1 with 100ng/ml PMA. It might be possible that 0.5 million cells for 24 well plate are to high with 25ng/ml PMA. Either reduce the number of cells (0.2 million) or try to use 100ng/ml of PMA. I never faced such cell loss problem.

all the best...