I am currently trying to differentiate U-937 cells into a macrophage like phenotype using PMA. I am currently using 800,000 cells in a mL of RPMI-1640 supplemented with 100ug/mL of PMA. After 48 hours the cells do tend to clump up more but they do not present as macrophages and will not adhere to the plate. Does anyone have any advice?
May I venture the comment that perhaps you should be using a PMA dose in ng/mL range, not ug/mL?
I used THP-1 and differentiation is sometimes performed. The dose is actually 5 ng/mL, validated in several papers.
http://www.ncbi.nlm.nih.gov/pubmed/17334670
As for U937, there is already an existing RG thread on similar topic.
https://www.researchgate.net/post/What_are_the_optimal_conditions_for_differentiation_of_U937_cell_line_in_macrophages
A paper providing evidence on protein changes after differentiation:
http://www.lrjournal.com/article/S0145-2126(10)00383-8/pdf
The method was 10 ng/mL PMA, 72 h.
http://www.sciencedirect.com.eproxy2.lib.hku.hk/science/article/pii/S0145212610003838
With a protocol from another paper:
P. Stöckbauer, V. Malasková, J. Soucek, V. Chudomel
Differentiation of human myeloid leukemia cell lines induced by tumor-promoting phorbol ester (TPA). I. Changes of the morphology, cytochemistry and the surface differentiation antigens analyzed with monoclonal antibodies
Neoplasma, 30 (1983), pp. 257–272
Yes 100ng/ml PMA is optimal.... and your cell density is too high. 50,000 cells-200,000 cells/ml is the range for a 24 hr duration for this 100ng/ml dose.
Dear Corey
We also tried to differentiate U937 towards macrophages using PMA. We used different protocols: stimulate cells with 30ng/ml for 3 days and same dose for 3 days but change medium and let them differentiate for additional 3 days. Most changes are seen with the latter protocol, however, although there are changes detectable on molecular basis (FACS, PCR) U937 did not become adherent. With THP1 it works perfect.
We use 50nM PMA, seeding density 200,000 cells/mL and incubate over night. The U937 cells adhere nicely to the plate...
Good luck!
I have not done with U937 cells but while trying to read about the differentiation of THP1 cells to macrophages, I have read in few articles that 50-100nM PMA treatment for 48 hrs may allow the U937 cells to differentiate and adhere on plate.
http://www.hindawi.com/journals/bmri/2014/683068/
They stimulate 106 cells with 200 nM PMA for 72 hours. The adherent cells were further cultured for 5 days in absence of PMA.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008668
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