Dear Asha!

Please you look at this article:

Article Toll-like receptor 4 signaling: A common pathway for interac...

RAW-Blue™ cells were derived from the murine RAW 264.7 macrophages with chromosomal integration of a SEAP reporter construct inducible by NF-κB. RAW-Blue™ cells express many pattern-recognition receptors (PRRs), including all toll-like receptors (TLRs) except for TLR5, but with TLR4 and TLR8 expressed at the highest levels. However, TLR8 is endosomal and a different ligand activates it from TLR4. Thus, RAW-Blue™ cells are useful as TLR reporter cells for inducing NF-κB, with subsequent release of SEAP after treatment with appropriate TLR-specific ligands, including ROS for TLR4 [23]. Therefore, we used RAW-Blue™ cells with the same read-out metric as HEK-Blue mTLR4 cells to affirm that the synergistic/additive effects of oxidants with HMGB1 can also occur in cells that express multiple TLRs.

Both HEK and RAW-Blue cells were grown in a 37 °C, 100 % humidified incubator in Dulbecco’s Modified Eagle’s Medium (DMEM, 4.5 g of glucose/L) without pyruvate but supplemented with 2 mM L-glutamine, 10 % (v/v) fetal bovine serum (FBS), 50 units/ml penicillin, 50 μg/ml streptomycin and 100 μg/ml Normocin™. HEK-Blue-Null1-v, HEK-Blue mTLR4 and RAW-Blue cells were maintained in growth medium supplemented with Zeocin.

Fig. 9The effect of prooxidants or HMGB1 alone and /or in combination on SEAP release in RAW-Blue™ macrophage cells that express multiple TLRs (except TLR5)

RAW-Blue™ cells are useful as TLR reporter cells for induction of NF-κB, with a subsequent release of SEAP after treatment with appropriate TLR-specific ligands, including ROS for TLR4 [23]. The QUANTI-Blue™ detection system was used to provide an easy and rapid means to quantify SEAP released into the culture media (based on the extent of TLR4 stimulation). *p ≤ 0.01 and +p ≤ 0.001 with n = 3 independent experiments conducted in duplicate.

Article Detection of Innate Immune Response Modulating Impurities in...

Fig 3Limit of detection to PRR ligands in PBMC and monocyte/macrophage cell lines.

PBMC, RAW-BLUE, THP-1 and MM6 cells were stimulated with the indicated concentration of (A-D) Endotoxin, Pam3CSK4 and FSL-1, (E-H) Poly I:C and Flagellin, (I-K) Imiquimod, CLO75 and CpG (L-O) zymosan and MDP. Levels of IL-8 mRNA (relative expression vs media), NF-κB activation (OD 620) and luciferase activity (RLU) were measured as described in materials and methods. All samples were tested in triplicate. Data points represent mean ± SD. (*p

Dear Alexander , Thank you so much for the article.

May I ask, I read some articles about positive controls drugs (tereparatide injection) test with RAW Blue cell line. Will those drugs stimulate cytokines expression or any Optical density in the assay?

My thought, is these drugs should inhibit cytokines release and OD should match with blank? Am I right?. Or high concentration of drugs can kill the Raw Blue cells?

Kindly share your thought....