THP1-NLRC4 cells

NLRC4 inflammasome test cell line

THP1-NLRC4 cells are derived from the THP1 human monocytic cell line, which represents the most commonly used model cell line to study inflammasome activation.

THP1-NLRC4 cells stably overexpress NLRC4 and naturally express TLR5. Stimulation of these cells with flagellin triggers TLR5 signaling leading to NF-κB activation and the production of pro-IL1β. Once in the cytosol, flagellin induces the formation of the NLRC4 inflammasome resulting in the activation of caspase-1 and the release of IL-1β.

Levels of IL-1β secreted into the supernatant of THP-1 cells can be monitored using the HEK-Blue™ KD-TLR5 cell line.

THP1-NLRC4 and HEK-Blue™ KD-TLR5 cells were co-cultured and stimulated with increasing concentrations of ultrapure flagellin from S. typhimurium (FLA-ST UP), B. subtilis (FLA-BS UP) or P. aeruginosa (FLA-PA UP), or monosodium urate (MSU, an NLRP3 inflammasome inducer which requires prior priming of THP-1 cells).

After 24h incubation, IL-1β-induced NF-κB activation was assessed by measuring the levels of SEAP using the QUANTI-Blue™ assay. When cultured alone, HEK-Blue™ KD-TLR5 cells do not respond to flagellin.

Specifications

Antibiotic resistance: Blasticidin

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin™, Pen-Strep (50 U/ml-50 μg/ml)

Quality control:

• Expression of NLRC4 was confirmed by qRT-PCR and a functionality assay using inflammasome inducers.
• THP1-NLRC4 cells are guaranteed mycoplasma-free.

Contents

• 1 vial of THP1-NLRC4 cells (3-7 x 10e6 cells) in Freezing Medium.
• 1 ml Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria and fungi.
• 100 μl Blasticidin (10 mg/ml).

Shipped on dry ice

Description

Application

THP1-NLRC4 cells are designed to study the activation of the NLRC4 inflammasome.
Mature IL-1β can be detected by Western blot, ELISA, or a cell-based assay.
InvivoGen has developed HEK-Blue™ KD-TLR5 cells, a reporter cell line designed to monitor bioactive IL-1β secreted by THP-1 cells upon flagellin-induced NLRC4 activation.
HEK-Blue™ KD-TLR5 cells are derived from the HEK293 cell line, which endogenously expresses TLR5 and the IL-1β receptor (IL-1R). This cell line features an NF-kB-inducible SEAP reporter gene and was engineered to knock-down the expression of TLR5 to avoid activation of NF-kB upon flagellin-induced TLR5 stimulation. The knockdown of TLR5 permits the analysis of flagellin specifically for its NLRC4 stimulating activity. Binding of IL-1β to IL-1R initiates a signaling cascade leading to the activation of NF-kB and the subsequent production of SEAP.
Detection of SEAP in the supernatant of HEK-Blue™ KD-TLR5 cells can be readily assessed using the QUANTI-Blue™ assay. Levels of IL-1β secreted into the supernatant of THP-1 cells can be monitored using the HEK-Blue™ KD-TLR5 cell line.
THP1-NLRC4 and HEK-Blue™ KD-TLR5 cell lines can be used sequentially (option 1) or co-cultured to save time (option 2).

THP-1/HEK-Blue™ IL-1β Assay :