Anti-inflammatory activity in RAW 264.7 macrophage cell line
Inflammation is a complex process that has been implicated with serious illnesses such as cancer, rheumatoid arthritis, diabetes, obesity, fatty liver, asthma, microbial infection, and Alzheimer’s disease. There has been a lack of effective anti-inflammatory agents that combat these pathological effects of inflammation with few side effects.
In the search of promising and effective anti-inflammatory agents, RAW 264.7 cell line model represents a powerful cell-based tool to investigate the anti-inflammatory potency of natural extracts, fractions, and/or isolated and synthetic compounds. This cell line is macrophage-like and is inducible for common mammalian pro-inflammatory cytokines such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), Cycloxygenase 2 (Cox-II), interleukins (eg, iL-6), and Tumor necrosis factor alpha (TNF-α).
Principle of the model
To mimic inflammation, the RAW264.7 model employs lipopolysaccharides (LPS) as it constitutes the chemical structure of many pathogenic bacteria, fungi, and viruses that cause inflammation. Thereby, LPS can induce these inflammatory pathways such as MAPKs and NFkB pathways via the Toll-like receptors in these stimulated cells. The common markers to measure in these types of assays are nitric oxide production, iNOS protein expression (Western blotting),IL-6 secretion, etc.
The inhibitory potency of the candidate test samples against the LPS-stimulated production of these pro-inflammatory cytokines is a good evidence for the promising use of these candidates as anti-inflammatory agents.
Figure-1 Schematic diagram for the inflammatory pathways in LPS-stimulated macrophage.
For each sample you need to carry either of protocols 1 or 2, then choose further readings from protocols 3 to 5.
|Protocol number||Assay name||Output|
|1||Griess colorimetric determination of NO inhibition in LPS-induced RAW 264.7 macrophages-two sample concentrations screening||% NO inhibition for 2 concentrations of the sample|
|2||Griess colorimetric determination of NO inhibition in LPS-induced RAW 264.7 macrophages-Concentration-dependent effect (IC50) for 1 sample||IC50 VALUE FOR NITRIC OXIDE inhibition for 1 SAMPLE|
|3||Anti-inflammatory testing by Western blotting (Protein expression of the inflammation marker inducible nitric oxide synthase, iNOS)-One sample with 2 screening concentrations on a single blot||Western blot showing iNOS and B-actin Expression for required controls and the tested 2 sample concentrations|
|4||Anti-inflammatory testing by Western blotting (Protein expression of the inflammation marker inducible nitric oxide synthase, iNOS)-Two samples with 2 screening concentrations each on a single blot||Western blot showing iNOS and B-actin Expression for required controls and the 2 tested samples|
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