I have differentiated THP1 monocytes into macrophage using 5 ng/ml PMA for 48 hrs. Then after washing twice with DPBS i have kept the diffrerentiated cells for 3 hrs in serum free media. Then I have added 100 ng/ml and 500 ng/ml LPS into the serum free media. I have harvested cells at 2 hrs and 6 hrs of LPS treatment but none of them were able to produce significant change in Cytokine gene activation (TNFa, COX2, TGFb etc) wrt diferentiated control sample. Plz help me out. how should i modify my protocol?
Thanks! I have used low serum (0.5%, heat inactivated) for LPS induction also but didnt get significant change in cytokine profile. Should i increase the serum conc for the treatment or should i use non heat inactivated serum for this purpose?
0.5% serum is too low. Use 5-10%. Also, check your cells for mycoplasma ( you may look at my article in PLOS One in 2010). Agree with Alexander that PMA may already activate cells and you are unable to see any additional activation.
Thanks Prof. Gavrilin. Answers were really resourceful. Your article on PLOS One is extremely helpful. I think I will be able to troubleshoot.
Hello, the serum level must be at 10% and the PMA concentration should be at 300ng/ml during 3h (enough for differentiation). Furthermore, I think that you should harvest your cell at 24h to detect properly TNFa, COX2, TGFb secretion. Finally, THP-1 cell do not express CD14 initialy but with PMA or Vit D3, CD14 is upregulated. Thus, have a look on CD14 expression on differentiated THP-1 cells to be sure.
According to my protocol, once monocytes are differentiated, you have to maintain them in serum free media. Resting phase after differentiation is important. Then 18-24 h incubation with LPS will induce sufficient amount of cytokines (TNF-a, IL-6, PGE2 etc). I think you are not giving enough incubation time with LPS.
Dear Anindhya, I agreed to most above. You should use 10% heat inactivated serum for growth of the cell. I wish to add one or two more point here-
1. I think you add too much of LPS, and for LPS induction effect u can check nitrite generation culture after 18 hrs by griess assay.
2. If too much of LPS is used, your cells can be LPS tolerant and will not respond to LPS anymore, this is called anergy.
Thank you Satvir Sekhon and Debanjan Mukhopadhyay for your reply. Your practical knowledge is really helped me to understand better.
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