I am working on a project that requires stimulating RAW 264.7 NF-kB macrophage and readout my signal by luciferase assay. Although all concentrations tested (10, 50, 100ng/mL) after 24 hours enhanced the NF-kB signalling the 50ng/mL appears to be slightly robust than the 100ng/mL and I unexpectedly observed no dose reponses?
Is there something that could account for such observation?
I wonder if you are seeing negative feedback in NF-kB at the high LPS concentration.
https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.00705/full
https://www.spandidos-publications.com/or
I agree with Ellen A G Chernoff .
In addition to the effect of LPS concentration, timing is key in capturing relevant levels of downstream actors (i.e. NF-kB in your study) before negative feedback kicks in.
Check out the findings on the abstract of this lab at 24hr vs 10 or 16 hrs
Article NFkB activation and cytokine output in LPS‐treated RAW 264.7...
This article Article LPS-stimulated NF- κ B p65 dynamic response marks the initia...
If you are looking for a consistent dose-dependent response from 10-100 ng/mL, perhaps, try reducing incubation time after LPS treatment. Notice the short incubation time used for 100 ng/mL LPS in the early days of elucidating the pro-inflammatory and transcriptional role of NF-kB. Article Xie Q-W, Kashiwabara Y, Nathan C. Role of transcription fact...
Otherwise, the drop you are observing between 50 and 100 ng/mL seems to indicate negative feedback to LPS stimulation- which is well-established. The article titled "Lipopolysaccharide Induces Hyporesponsiveness to Its Own Action in RAW 264.7 Cells" by G.D. Virca et al, 1989. shows good evidence for this negative feedback with a range of concentration and incubation time.
Good luck!
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs