Hi everyone,
I am working with THP-1 for my PhD project and I am having problems after the treatment with PMA.
I follow this protocol:
1. Seed the cells in 6-well, density 3x10^5 cells/ml
2. After 24h from seeding, add 10 ng/ml of PMA (24h treatment)
3. After 24h from the PMA treatment, change the media and leave the cells to rest for 72h
4. Following this resting period, to obtain M1 I treat the cells with 100 ng/ml of LPS and 20 ng/ml IFN-gamma, instead for M2 with 20 ng/ml of IL-4 and 20 ng/ml of IL-13
The problem is that, at the end of the resting period (72h), most of the cells are in suspension and the ones adherent have an abnormal phenotype, like they are dying.
As culture media, I use RPMI 1640 + 10% FBS no heat inactivated + 1% Pen/Strep + 1% L-Glut. Would anyone have any advice for me to solve this problem?
Try to increase PMA concetration (25ng/ml) and leave them 48hrs. Maybe your cells are not attached enough. I am also using heat inactivated FBS without P/S and it works good for me.
You can use several different concentrations 10, 20 30 ng/ml and different activation time 24, 48 hrs and see the results. Did you use the cold ice to detach the cells?
I tried to increase the concentration of PMA and actually, the adhesion improved (ideal starting from 50 ng/ml) but at this point, another problem arose because I couldn’t get a good polarization of cells. Indeed, the M0 macrophages over-express markers of the M1 phenotype.
About 25 ng/ml PMA, I didn't check for the polarization but I'm going to try, maybe this is the right compromise between differentiation and polarization.
Erjola Rapushi you're using heat-inactivated FBS, aren't you? I read that for THP-1 is important to work with "no heat-inactivated FBS". Another hypothesis could be that the activated complement proteins in my FBS are affecting the viability of the macrophages. I can try changing the media (RPMI + heat-inactivated FBS) following the treatment, what do you think?
I am using heat inactivated during THP1 culture. During the resting period (48hrs) i am using 10% heat inactivated FBS and it works for me.
Hi Giorgia,
You can go with 500,000 to 1m cells per well in 6 wells plate. If you have been growing these cells (not frozen), there's no need to seed them for 24h before PMA addition. Just seed your cells alongside PMA (at 80ng/ml or 100ng/ml _works very well for me) for 24h. After 24h replace with PMA free complete RPMI medium for addition 24h. Remember you are working with monocyte-macrophages, I think the high M1 phenotype you experienced for the M0 cells after 72h rest period is because of dying cells (detached cells) acting like DAMPs to further activate your cells. Make sure you wash your cells about 3 times with pre-warmed medium (to remove any dead cell) before any simulation.