I want to perform infection studies in singularly polarized macrophages i.e. either M1 or M2. How do I maintain THP-1 or primary macrophages in singularly polarized manner?
Hi Durgesh, you can polarize human monocytes into either M1 (with IFNg and LPS) or M2a (IL-13 and IL-4). Be aware though that there are different subsets of M2 (a, b and c) that are obtained with different cytokines and/or administration of immune complexes.
If you want to obtain macrophages from tissue or peripheral blood you probably need to do sorting. I don't know how feasible this is since their number is likely to be relatively low.
Good luck,
I agree with Marc's answer, and additionally, for THP-1, there is an optional way to maturate the monocytes into macrophages by adding PMA prior to cytokines for polarisation (you could try 24 or 48 hours). Some researches are mentioning vitamin A, but I myself tried both and decided PMA gives better maturation (CD68+ by FACS). Good luck!
Macrophage responses to activating conditions are dynamic. There are genes that change expression rapidly but transiently. Other genes may change rapidly and maintain their elevated or repressed expression levels. A third set of genes may change in a delayed manner based on indirect mechanisms such as LPS inducing interferon beta, which then causes changes though pathways that are not directly downstream of LPS/TLR4 pathway such as the Jak/STAT pathway. I have seen that gene expression profiles tend to stabilize after a day or two of continuous activating conditions compared to the changes that occur during the first few hours.
http://journal.frontiersin.org/article/10.3389/fimmu.2015.00253/abstract
Hi,
Treat THP-1 with PMA for 24 hrs and rest it for atleast three days. Stimulate then for 24 hrs with IFN-gamma to get M1 population and with IL-4 for M2.
I have'nt tried this with primary culture.
Hope this helps.
Kamal
I agree with that you should treat cells with PMA first.
I use 20ng/ml media of IFN for M1 and 10mg/ml media IL4+IL13 for the M2. I noticed that the best M2 activation is obtained after 48h treatment, whereas M1 activation is significant within 24h.
What is your model for infection? Just asking because LPS has been shown to result in a somewhat different M1 phenotype.
We use 10ng/mL M-CSF (95-99% 'M2') and 20ng/mL GM-CSF (75-85% 'M1') straight on monocytes or bone marrow suspensions. We consider this more 'polarised' and you can use the same treatment on both eg TLR ligands.
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