THP-1 Cells differentiation - THP-1 cells differentiated using IFN-gamma/LPS (Jun/28/2010 )

I'm trying to set up a few in vitro assays using THP-1 cells that I have been growing in suspension. All the literature on the subject says to plate the cells on poly-d-lysine coated plates and then add IFN-gamma and LPS to induce the cells to differentiate into a macrophage phenotype. These plates are no where in stock in Australia, which leads me with a problem. There are a few other types of plates that have a treated surface which have been recommended to me, but I am not sure if they will work (e.g. CellBind, CC2, etc). Does anyone know if there is any alternative to poly-d-lysine coated plates for THP-1 cells? Or even how well differentiated THP-1 cells adhere to different surfaces?


I am working on P2X7 receptors, if that helps, and am going to be stimulating the adherent cells with ATP with or without an antagonist after applying various dyes (Yo-Pro or Fluo-4 AM). Thanks for any suggestions!

Hi,

I worked with THP-1 macrophages for a couple of years and never used poly-d-lysine coated plates. I simply used the conventional 12 well plates without any problems. You also mentioend that you were told to induce the macrophage phenotype by hitting them with LPS-IFNg combination. This would induce a classically activated phenotype and not a naive phenotype. If you wanted to induce a naive type macrophage hit 250,000 cells/ml with 20 nM PMA for 2-3 days.

What is the difference between a classical phenotype and a naive phenotype? Just yesterday, I used PMA (0.5 uM) to differentiate and LPS (10 ng/ml) to stimulate the cells. I haven't run through the entire assay yet so I'm unsure what the results will be, but the coated v. uncoated plates didn't seem to make a difference in morphology changes or how well the cells stuck down (both seemed to come out of suspension and become adherent relatively well once treated with PMA).

I have read that the coating on the plates helps to upregulate various membrane proteins and I'm not sure how this will affect my assays. Any suggestions? Thanks for the help!

You don't need coated plates. I have induced THP1s in uncoated plates as well as in just regular NUNC T75 flasks. They adhere just fine, within hours after stimulation with PMA. I don't think I have ever seen coated plates mentioned before.

Corina on Jul 8 2010, 01:04 AM said:

Good question. It's a dynamic paradigm with different trains of thought depending on who you are talking to. I suggest to pour through the literature and read about macrophage differentiation (polarization) ie. Classically activated vs Alternatively activated macrophages. Also M1 vs M2 (M2a, M2b, M2c). The problems with these classifications when working in vitro though, are their heavy plasticity and the fact that their markers can be temporally dependent on your various induction cues.


I'm no immunologist by any means, and I am pretty much learning as I go working with these guys. Depending on how in depth you need to go into this polarization paradigm, be ready to read lots of papers.

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

Hi Anula, it would be best to store PMA stock at -20C, and thaw once only when adding to media. It is also light sensitive, according to the Sigma product sheet (http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/p8139pis.Par.0001.File.tmp/p8139pis.pdf)

Thank you lamaksha!

Could anybody tell me how long I should keep my THP-1 in fresh RPMI medium after treating with PMA? 3 days will be ok??

Anula on Wed Mar 21 08:12:08 2012 said:

At certain concentrations, the cells will respond as macrophages within 2 hours. See the Fernandes-Alnemri 2007 and 2009 papers about the pyroptosome for more info on that.

From personal experience, 1-3 days (longer being better) is the norm for getting proper morphology of differentiate macrophages. You can change the medium as needed without the addition of PMA after the first day and it should still be OK.