I am trying to assay the IL-1b from culture supernatant of mouse macrophage cell line but I'm not getting results. I am getting results with standard but not with samples, though I don't know why?
2x10^5 cells were plated and stimulated with 500 ng/ml LPS for 24 hrs, but I don't see a difference between the untreated and treated plates. I am using Invitrogens mouse IL-1b ELISA kit for this purpose.
You need add ATP one hour before harvesting supernatant, in order to activate inflammasome-mediated cleavage of pro-IL-1b.
check if your LPS is working and mycoplasma contamination in your culture. Also its a good idea to starve your cells before stimulation for 6-12 h with 1% FCS containing media for better stimulation.
I am getting good results for TNF-a from same sample, why this is happening?
You need add ATP one hour before harvesting supernatant, in order to activate inflammasome-mediated cleavage of pro-IL-1b.
Hi Sagar,
I do not have experience with cell culture and IL-1 but in vivo IL-1 can be an early response cytokine, ie 1hr after adding toxin. Should you perhaps check at different timepoints? Just an idea.
Hi Sagar, Mingjun is spot on. You need a secondary activation in the form of ATP . I would stimulate bone marrow derived. macrophages for 4hr with 100 ng/ml LPS and then 5mM ATP for an hour. Works very well and gives me IL-1B in the range of 1-3 ng/ml. Hope this helps.
You could also try to combine LPS with IFNgamma, this combonation enhances the production of pro-inflammatory mediators, due to the fact that IFNgamma is a coactivator of the LPS response. Please have a look in my publication from 2012...
- Badges
- Science topic