Kindly suggest me optimized PMA concentration that will work well for differentiating human monocytes THP1 into macrophages. I am facing problem with PMA concentration which is not helping THP1 cells to attach, using 100ng PMA. What can be better way to achieve this?
100ng/mL should be more than enough to differentiate THP-1. I have done with 40ng/mL and it worked. Is your PMA working? Was is properly stored? Are you sure your cell is THP-1? (many labs have problems with cell line misidentification)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96928/
http://www.ncbi.nlm.nih.gov/pubmed/17334670
20 ng/ml was enough to differenciate THP-1 cells.
Be sure you re ading the right suppléments, and also working with fresh aliquots of THP-1 cells not more than 10-15 passages.
https://www.researchgate.net/publication/5932602_Mycobacterium_bovis_Bacillus_Calmette-Guerin_Secreting_Active_Cathepsin_S_Stimulates_Expression_of_Mature_MHC_Class_II_Molecules_and_Antigen_Presentation_in_Human_Macrophages
Article Mycobacterium bovis Bacillus Calmette-Guerin Secreting Activ...
16-20 nM for 1-3h of treatment is enough to differentiate THP-1 cells into macrophage-like cells if your cells come from a fresh cell culture. If you've been growing them for long times (more than 12-16 weeks), then you will need about 48h and lower doses of TPA to get your cells adhered. Be sure that your THP-1 cells grow in suspension (aprox 99%) before treatment and that your cells are not over-growed (less than 5x10exp5 cells/ml). RPMI 1640 + 5% FBS + kana is a good growing médium for them. You can test your cells for C3 anc Fc expression. The drug can be easily degraded. Take care of that.
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