What kind of cells are using?

Could be a lot of different reasons. What cells and what time point are you collecting the sups at? If using macrophages/monocytes, try using less LPS. It is usually used at the 10-100 ng/mL. Collect sups at 24 hours or later. The longer you leave the cells with the stimulus, the better your chances. Also try adding the intermediate step of converting your nitrates to nitrites. If you are still having trouble, make sure your LPS works by looking at cytokine production or try one of the other TLR agonists.

Hello

Nawzat, we are using RAW 264.7.

Hello, Julie Joseph.

I use RAW 264.7 and let it incubate for 24 hours. After this time collect the supernatant. Thanks for the suggestions.