Hello

I only worked on murine BMDMs, not on J774. However, the LPS used is very important as not all LPS reached to induce inflammatory responses. I used LPS (L3024) form Sigma-Aldrich.

I think that with the concentrations you used, you would be able to see IL1beta mRNA.

The other point is the possibility to need a second signal, notably for inflammasome activation and IL-1beta production.

Hope this can help you.

I am not expert in this area but, I did publish one paper using the activation of macrophages as below:

U937 cells (1million/ml) treated in serum free medium for 24h, washed with serum free medium once, activated with 12.5ug/ml LPS and 60ng/ml PMA for 1h

I will recommend you to discuss with technical advisor from Sigma Chemical company since your cell line and LPS are from Sigma. They will be able to give the right direction or alternative suggestions,

Good luck

Hey Natalia,

we normally work in our lab with peritoneal macrophages (PM) and bone marrow derived macrophages (BMDM) and at one point shortly with RAW macrophages. We treat all of them with 5 µg/ml LPS f.c. in the well.

The general experience is that in vitro differentiated macrophages or macrophage-like cell lines react much slower not only to LPS but also to bacterial infection. It takes 24 h to detect cytokine levels in supernatants with BMDM or RAW (either with LPS or after bacterial infection) but only 5 h for PM or macrophages diffentiated from human blood.

I personally think that an ELISA for IL-6, TNF, IL1-ß and other cyto/chemokines is best suited for this analysis. Western Blot only detects cytokine protein levels in the cells but most cytokines are build for secretion and mRNA expression does not equal a functional protein (mRNA can be stored by the cell without protein build).

So I would recomend that you seed out your macrophages with 150,000-300,000 cells per well in duplicates for each time point (lets say 0h, 5h, 24h, 48h). At each time point take the supernatant and store it at -20°C and analyze with an ELISA later.

I know ELISAs are expensive, but intracelluar protein or mRNA levels do not represent secreted cytokines and thats it what cytokines are for in the body.

For further information on stimulation and cytokine levels of different marophages you can have a look at:

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO

February 2019 Science Signaling

I hope that helps you.

All the best,

Marc Herb

I agree to the aforementioned comment.