There is an increase in NO production and increase in phagocytosis (increased uptake of Neutral Red) but Alamar Blue assay shows a drop in cell numbers. Any explanation?
We have worked with 1ug/mL of LPS to activate RAW cells and had similar problems. At 1ug/mL it did increase NO production and CD40 expression etc, however the viability was lower than control. later on we tested lower concentrations and figured out that,0.125ug/mL works good enough. it didn't really induce a proliferative response, but at least it didn't affect the viability and could induce the responses we were looking for. I cant think of another reason other than the concentration of LPS.
However, even i am curious to know why macrophage fail to exert a proliferative response to LPS stimuli. plz let us know if u mange to tackle the issue.
all the best!
I had a look at some papers and found one which was very vague, but mentioned when they are activated they stop proliferating, (but is not toxicity), but I'm trying to understand why this happens, but it's good to know I'm not the only one who experienced this.
As a myeloid cell matures it looses its proliferation capacity. Macrophages are quite mature cells and they hardly proliferate. As RAW cells are cell line they retain capacity to proliferate. However antigenic stimulation (i.e. LPS) induces both activation and maturation of myeloid cells. So the answer is "LPS promotes the maturation of your cell line thus it stops doubling". If you culture them longer periods with LPS, these cells will face apoptotic death following maturation.
Thanks , but are macrophages not already matured or is this a characteristic maintained in this cell line, also do you have any references where I can read more about this. Thank you so much
NO is a gas that freely diffuses in all directions. If macs (or mac cell lines) cause more than about 7uM to accumulate in a dish, the macs suffer/perish. They are not resistant to NO killing - they "die in the battle" Good luck. Charlie
Well, one may say that these cells looese some of their maturity characteristics as they are established as a continuous cell line. I have some experience (can see one of my publications) with J7 macrophage cell line. I think you need to find some review papers on monocyte/macrophage maturation/differentiation. Good luck with your research...
Since it is established that the synthesis of NO is affected by the LPS concentration, certainly increased production of the NO beyond a certain threshold affects proliferation. One would consider trying diferent protocols to determine the most favourable LPS concentration.
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