I want to test a reagent and see if it has an inflammatory effect. Therefore I treated the Cells 5h with 1 µg/ml LPS as a positive control or left them untreated. However the untreated cells showed in both cases higher concentration of the cytokines than the LPS stimulation. What could possibly go wrong? I even leave the cells for 24h after media change and add just the stimulation to prevent stress.
This happens mostly due to receptor internalisation, which method did you use to measure the levels of ILS
Have you tried reducing the serum/growth factors in the medium 24hrs before stimulation? This should drop any background inflammation levels allowing a clear induction of IL-6/8 with LPS.
Also are the untreated control levels of IL-6 mid-range on the standard curve or are you actually looking at very low IL-6 levels across all samples? If this is the case then your LPS may not be stimulating as you expect.
Karthika Karunakaran I used R&D Duo Set for ELISA in both cases!
Mark Ian Fowler I havent tried yet, thanks for the advice! I will definitely try! The problem was , that for IL6 there was no difference between the unstimulated control and the treatment (all between 60 and 80 pg/ml) and IL8 had 1200 pg/ml whereas the stimulations were in between 800 and 900.
I will report if it was successful, thank you very much
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus