I am stimulating THP-1 cells by PMA to differentiate them into macrophages. I used different conc. of PMA ranging from 8nanoM to 0.2microM. the duration of stimulation was: Day 1, 2, 3. In all conc. and all periods, only 30-35% of harvested cells were live cells. I am really concerned about this viability issue. Could you suggest anything to improve the viability of PMA-stimulated THP-1 cells?
Cell seeded: 1x10e6 cells/well in 6-well plate.
Cell harvest: Following PMA stimulation, I harvested cells by cells scrapper or leaving cells on ice for 20min. But later did not work.
FACS data showed that Cells express CD68 but there are lots dead cells (7-AAD).
How did you harvest the cells? THP-1 cells become adherent during differentiation, maybe you need to adjust the harvesting process.
Harvesting differentiated THP1 cells is very difficult. They stick down to plastic quite well and you do get a lot of cell death after that. Do you really need to harvest them. Can't you use them as stuck down?
Hi Abdullah,
I use PMA at 5ng/ml for 18-24 hours to differentiate my cells and have found that this is sufficient for differentiation without causing non-intentional upregulation of molecular pathways.
I tend to wash the cells with pre-warmed PBS (without calcium or magnesium) (to remove non-viable cells and debris - do you do this?) and either:
- add Trizol directly to the cells after differentiation for RNA analysis or
- add RPMI and scrape the cells in order to harvest. I spin the cells at 300xg/10mins and then carry out protein extraction. Are you looking at intracellular or extracellular proteins?
You may also want to try adding ICE COLD PBS+5mM EDTA (instead of RPMI) to encourage detachment from the plate. You may be able to do this without scraping the cells, if you leave the samples on ice for about 5 mins.
You will get some cell death upon the harvesting process as, as Nilesh mentioned, it can be difficult to harvest adherent cells and the scraping procedure will definitely cause some damage but if you are not looking at extracellular proteins and you keep the samples ON ICE (to prevent protein phosphorylation) during cell harvest, this should not be an issue.
I hope this helps,
José
Forgot to mention, if attempting to harvest cells (with or without scraping), always check the plate (before and after harvest) under an inverted microscope to ensure the majority of cells have gone into suspension and very few are left on the plate. Sometimes scraping is necessary.
The main question is: how do you look at your cell viability ? MTT ? Cell count ? Anything else ? Without these information it is not quite possible to answer, because as stated, if you scrap these cells, you will easily get more than 50% cell death, and this is not related to PMA treatment.
Also Jose, did you check (for example by flow cytometry with CD68) that your macrophages are fully mature with your treatment ? Personally I use 50ng/mL for 72h, because this is at this concentration that my THP-1 cells exhibit "true" macrophage-like patterns. Below these concentration and time, they still attach, but lack many macrophage feature, and therefore lack relevance.
Hi Alexis, I used CD36 expression to determine cell differentiation. These cells are definitely more active than undifferentiated THP-1 (after LPS stimulation) but I will look at CD68 using FACS. Thank you for the suggestion.
You can check this paper for more information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC26222/
They treat with 100 nM (= 62.5 ng/mL) for 3 days
Hi, scrapping is not a good idea and if you only check cell viability of differentiated cells after scrapping by flow cytometry 7-AAD is quite normal having 50% of dead cells. As suggested, perhaps MTT assay can help to determine cell viability of the THP-1 cells without detaching them, and then you can compare with your data to know if the loss of cell viability could arise from PMA treatment or scrapping. The differentiation degree could be checked in the living cells after scrapping, and the final assays could be done directly over the wells with differentiated THP1 cells without scrapping. In addition, having healthy THP1 cells avoiding overgrowth (>million cells/mL) and checking mycoplasma infection (THP1 cells are very susceptible and after many passes you should come back to a first-pass vial) will help you to have viable cells and to avoid unspecific activation in resting conditions. Good luck!
If you really want to see the viability of the cells, just put trypan blue in your medium, (0.1% final concentration) , then wait 1-2 minutes, take the medium out , and watch the cells!!! I had the same problem and gave up trying to take them out.
Check details for differentiation of THP-1 cells in our paper in PLoS One.