as Cell line shouldn't the whole cells express the same receptors, I wonder why it's really small percentage of cells only express that, I have repeated the experiment many times and always get almost the same percentage of cells.
I have obtained THP1 from ATCC, for differentiation, I used PMA 100nM for 48 hours then resting time in PMA-free medium for 5 days.
If I understand the question correctly, you noted that only a fraction of PMA-treated THP-1 cells expressed detectable levels of CD14. This could be due to a heterogeneity in cell population. One can "clone" a cell population of interest and study the clonal population.
@Divaker , But regarding THP1 as a cell line, is there any articles revealed the cell heterogeneity is this cell line. (So that is why only very small fraction are expressing CD14 or CD36) ?
In our lab we also differentiate THP-1 cells in 100 nM PMA for 48 hours and rest overnight before performing experiments. I have noticed that if you wait five days as you are doing that the cells start to de-differentiate back into a monocyte like cell again. Is there a specific requirement for resting the cells five days before doing your experiment? I would consider checking the markers the day after you remove PMA and see if the percentages are higher then.
Also worth asking, did you recently purchase these cells from ATCC or have they been in your lab for some time? Cell lines that have been passaged a lot in a lab can drift in phenotype.
Adding PMA to your THP-1 differentiates them into macrophages which means down-regulation of CD14. Also you don't need to rest them for 5 days. I used to treat them with PMA for 24hrs and then put them on serum-free RPMI medium for another 24hrs. After this you can do experiments with them (LPS or oxLDL, etc).
In addition, I agree with Jared. If THP-1 cells have been passaged to often or when people let them become to confluent they change in their phenotype. The way you can see this is that in normal culture (no PMA or anything) these monocytes should not adhere to your flask. If they do throw these out and get a fresh vial from ATCC to do your experiments with.
Undiferentiated THP-1 cells do not express CD14, so either something in your culture cause their differentiation or you observe non-specific binding of anti-CD14 antibody. Low CD14 expression on PMA-differentiated cells is not surprising as monocyte-macrophage differentiation is associated with CD14 down-regulation.
@Szecepan u said that undifferentiated THP1 don't express CD14. Isn't CD14 is marker for monocytes?
which marker should be positive for THP1 ?
@Tarek Yehia
Check out page 11 on this document from BD biosciences. It has a nice figure showing what markers you should see for THP-1 monocytes and macrophages.
As with primary monocyte-derived macrophages this figure shows that CD11b and CD11c would be a good markers to distinguish between THP-1 monocytes and macrophages.
I hope this helps.
https://www.bdbiosciences.com/documents/BD_Multicolor_MonocyteMacrophageDiff_AppNote.pdf
The most straightforward sign of differentiation is that THP-1 cells start to adhere.
Indeed adhering cells are not monocytes anymore and if this happens it is a clear sign of differentiation. If you still want to do flow, consider scraping the cells gently and do not use any trypsin to detach them. Upon differentiation to macrophages HLA-DR is downregulated and CD40 and CD64 are both upregulated in my experience.
CD14 is a marker for both human primary classical and intermediate monocyte subsets, but is quite low on THP-1. Keep in mind THP-1 are immortalized leukemia cells and can therefore have different expression markers as the primary monocytes.
I came across a study reporting on culture conditions (i.e. concentration and time) of THP-1 which can affect their response to PMA. When THP-1 cells are cultured at high confluence for long, the culture becomes heterogeneous and is highly sensitive to PMA with increased of CD14 expression versus low confluence of THP-1 culture.
This article might give you a better insight to your question
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703650/
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