Quanti blue detection medium

CpG (free or conjugated) potency was evaluated using the RAW-Blue reporter cell line (Invivogen, raw-sp), derived from murine RAW 264.7 macrophages. Stimulation of the reporter cell line with CpG induces TLR9 signaling pathways leading to the activation of NF-κB and AP-1, and the subsequent production of secreted embryonic alkaline phosphatase (SEAP). RAW-Blue cells (1 × 10⁵ cells/well) were incubated with CpG, CpG conjugates, or PIP at various concentrations in 200 μL/well for 24h at 37°C in 5% CO₂ in DMEM + 10% heat-inactivated (HI) FBS + 1% P/S. After 24h, supernatant (50 μL/well) was transferred to wells of a new 96-well flat bottom plate containing warm QUANTI-Blue detection medium (150 μL/well; Invivogen) and incubated for 2h at 37°C before measuring absorbance at 625 nm (A625) by plate reader (SpectraMax i3x Multi-Mode microplate reader). Data are reported as fold change in NF-κB/AP-1 activity compared to the untreated control (A625 value of sample divided by the average A625 of untreated technical replicates). Each independent experiment had 3 technical replicates. Data represent the average of technical replicates in three independent experiments; error bars represent the SD of the independent experiments.

THP1-XBlue cells stably expressing an NF-κB–inducible and activating protein 1–inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene (Invivogen) were cultured in RPMI 1640 supplemented with 10% (v/v) FBS (Gibco), penicillin (100 U/ml) and streptomycin (100 U/ml) (PAA), Normocin (100 μg/ml; Invivogen), and blasticidine-S-hydrochloride (10 μg/ml; Sigma). To monitor the activation of NF-κB signaling, 1 × 10⁵ cells were stimulated with 0.5 μM FBG or LPS (0.5 ng/ml) for 1, 4, 8, or 24 hours, and the amount of secreted SEAP was measured by mixing 20 μl of the culture medium with 180 μl of QUANTI-Blue detection medium (Invivogen) and incubated for 2 hours at 37°C. Absorbance was measured at 620 nm with a FLUOstar Omega microplate reader (BMG LABTECH).