Does anyone use RAW264.7 or J774 to check the proinflammatory responses of certain stimulus? How much TNF-alpha is typically produced by resting cells for each cell line (~105 cells)?
Why do you think these cells should release TNF without stimulation? We don't see any release.
In the absence of LPS or other stimulation, the level of TNFa is undetectable by qPCR or ELISA in RAW264.7, THP1 or MAC cell lines.
I know the level will be very low but it will be detectable. Please check out this paper
1. "Tumor necrosis factor-α mRNA remains unstable and hypoadenylated upon stimulation of macrophages by lipopolysaccharides".
2. J Immunol. 2008 Oct 1;181(7):4461-70.
IFN-gamma-induced TNF-alpha expression is regulated by interferon regulatory factors 1 and 8 in mouse macrophages.
Vila-del Sol V1, Punzón C, Fresno M.
They have reported the expression level of TNF-alpha in untreated RAW cells around 100pg/ml.
Are you sure you FBS is endotoxin free? As a rule, we test every lot of FBS before buying on endotoxin by its potential ability to stimulate monocytes. We check expression levels of TNF, IL8 (KC) and IL1B by qPCR and ELISA. Not all FBS are equal.
Exactly. FBS can be a major source of endotoxin and RAW264.7 are ultra-sensitive.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA