2.3.2. HEK BlueTM LPS Detection Assay

The HEK BlueTM LPS detection assay, obtained from InvivoGen (San Diego, CA, USA), is a very simple, sensitive, and reliable assay to detect biologically active LPS. Briefly, 20 µL samples were added and a LPS standard curve ranging from 12.5 to 0 ng/mL was made. Endotoxin-free water was used as the diluent. hTLR4 cells were prepared at a concentration of 140,000 cells per mL and 180 µL were then added to the samples, incubated at 37 ◦C, 5% CO<sup>2</sup> for 16 h. Afterwards, NF-κB activation was measured in detection medium containing Quanti BlueTM by reading the absorbance at 650 nm. The remaining protocol was followed in accordance with the manufacturer's instructions.
