TLR4 activation assay

HEK-Blue hTLR4 cells (InvivoGen) were cultured in Dulbecco’s modified Eagle medium, high glucose/l-glutamine supplemented with 10% fetal bovine serum, penicillin (50 U ml⁻¹), streptomycin (50 mg ml⁻¹), and Normacin (100 μg ml⁻¹) at 37°C in a humidified incubator containing 5% CO₂. After reaching ~50 to 80% confluency, cells were plated into 96-well plates at a density of 1.4 × 10⁵ cells/ml in HEK-Blue detection medium (InvivoGen). Antigens were added at the following concentrations: purified protein (100 ng μl⁻¹) and total protein in lysate (100 ng μl⁻¹). Purified E. coli O55:B5 LPS (Sigma-Aldrich) and detoxified E. coli O55:B5 (Sigma-Aldrich) were added at 1.0 ng ml⁻¹ and served as positive and negative controls, respectively. Plates were incubated at 37°C, 5% CO₂ for 10 to 16 hours before measuring absorbance at 620 nm. Statistical significance was determined using paired t tests.