Detecting Immune Responses to Type III Secretion Systems

Abstract

Measuring immune responses against type III secretion and related molecules has been made easier with the advent of reporter cell lines. For example, the THP-1-XBlue and HEK-Blue cells from InvivoGen provide easy detection of gene activation under NF-kB and AP-1 control. In addition, many of these cells have been engineered to express specific pattern recognition receptors (PRRs) helping to elucidate activation pathways. In addition, the THP-1 parent cell line from ATCC provides a useful tool for evaluating cytokine release upon stimulation. This chapter will outline using X-blue reporter cell lines, setting up experiments to measure cytokine secretion, and general guidelines for cytokine quantification.

Role of CD44 in Regulating TLR2 Activation of Human Macrophages and Downstream Expression of Proinflammatory Cytokines

“…THP1-XBlue cells (Invivogen, USA) is a THP-1 monocyte cell line that stably expresses AP-1 and NFκB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon activation of TLR2, SEAP is secreted and its activity can be monitored using an alkaline phosphatase substrate (31). THP1-XBlue cells were cultured in RPMI 1640 media supplemented with 4.5 g/L glucose, 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL normocin and 2 mM L-glutamine.…”

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14

“…To evaluate CMP here, we selected the engineered TLR4 model HEK-Blue h TLR4 cell line (HEK-TLR4; Invivogen, San Diego, CA) and its TLR4-negative HEK parental cell line (HEK-Null) because of their overall consistency, availability, controlled TLR4 expression and predictable ligand responsiveness. The correlation between TLR4, NF-κB and SEAP in this reporter cell line has been described in detail in the literature [ 36 – 38 ]. HEK-Null cells lack TLR4, MD2 and CD-14, but contain the same NF-κB SEAP reporter as HEK-TLR4 cells and were used as a negative control throughout the study.…”

Synovial Fibrosis in Osteoarthritis of the Knee: Mechanism of Development and Potential Therapeutic Targets

“…Impact of HA and CD44-specific antibody treatments on TLR2 ligand induced nuclear factor kappa B (NFκB) nuclear translocation in THP-1 monocytes and macrophages THP1-XBlue cells (Invivogen, USA) is a THP-1 monocyte cell line that stably expresses AP-1 and NFκB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Upon activation of TLR2, SEAP is secreted and its activity can be monitored using an alkaline phosphatase substrate[133]. THP1-XBlue cells were cultured in RPMI 1640 media supplemented with 4.5 g/L glucose, 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL normocin and 2 mM L-glutamine.THP1-XBlue cells (50,000 cells per well in HEK detection media) (100 μL per well) were treated with Pam (5 ng/ml) in the absence or presence of HA (MW >950 kDa; R&D systems) at a final concentration of 250 and 500 μg/ml, an anti-CD44 antibody (2.5 μg/ml; Abcam) or an isotype control antibody (2.5 μg/ml; Abcam) for 24 hours followed by measuring the 630 nm absorbance.THP-1 macrophages (600,000 cells per well) were treated with Pam (5 ng/ml) in the absence or presence of HA (100, 250 and 500 μg/ml) for 1 hour followed by cell harvesting and nuclear protein extraction using a commercially available kit (Thermo Fisher Scientific, USA).…”