RAW-Blue™ Cells

Notification: Reference #raw-sp-av can only be ordered together with reference #raw-sp.

Mouse macrophage NF-κB-SEAP reporter cell line

RAW-Blue™ cells are designed to monitor the NF-kB and AP-1 responses upon PRR (pattern recognition receptor) stimulation. These cells are derived from the murine RAW 264.7 macrophages. They feature an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.
RAW 264.7-derived cells endogenously express many pattern-recognition receptors (PRRs), including toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and C-type lectin receptors (CLRs) (see figure).

Therefore, RAW-Blue™ cells can be used as:

• TLR reporter cells: they express all TLRs, except TLR5, at different levels
• NOD reporter cells: they express NOD2 and, to a lesser extent, NOD1
• RLR reporter cells: they express RIG-I and MDA-5
• CLR reporter cells: they express Dectin-1

The stimulation with specific agonists of these PRRs induces signaling pathways leading to the activation of NF-κB and AP-1, and the subsequent production of SEAP. Levels of SEAP can be easily monitored using QUANTI-Blue™ solution, a SEAP detection medium (see figure).

Key features:

• Verified expression profile of different PRRs 
• Functional validated with a selection of PRR ligands
• Readily monitoring of NF-kB or AP-1 activation by assessing the SEAP activities

Applications:

• Study of various PRRs in innate immunity
• Screening of novel PRR agonists or inhibitors

Figures

Expression profile of various PRRs in RAW-Blue™ cells. RT-PCR results show endogenous expression of Toll-like receptors TLRs (1-9), RIG-I, MDA-5, NOD1, NOD2 and Dectin-1 in RAW-Blue™ cells.

NF-κB responses in RAW-Blue™ cells to various PRR ligands. Cells were incubated with TLR, Dectin and NOD agonists: TLR2 (HKLM, 1x10⁸ cells/ml), TLR1/2 (Pam3CSK4, 1 µg/ml), TLR2/6 (FSL-1, 1 µg/ml), TLR3 (Poly(I:C) HMW, 100 μg/ml), TLR4 (LPS-EK, 10 μg/ml), TLR5 (FLA-ST Ultrapure (UP), 10 μg ml), TLR7 (CL075, 10 µg/ml), TLR9 (ODN1826, 10 μg/ ml), NOD1 (Tri-DAP, 10 μg/ml), NOD2 (MDP, 10 μg/ml) and Dectin-1 (Zymosan, 100 µg/ml). After 24h incubation, TLR and NOD stimulation was assessed by measuring the levels of SEAP using QUANTI-Blue™. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

Specifications

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™ 

Antibiotic resistance: Zeocin®

Quality control:

• Expression profile of TLRs (TLR1 to TLR9), RLRs (RIG-I and MDA-5), NODs (NOD1/2), and Dectin-1 was determined by RT-PCR and functional assays.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells, in a cryovial or shipping flask
• 1 ml of  Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)