Tlr reporter cell lines

A TLR reporter cell line system that monitored TLR activity using secreted embryonic alkaline phosphatase (SEAP) induced by NF-κB and AP-1 activation was used. HEK-Blue™ Detection (InvivoGen) reacts with SEAP to turn the medium color purple/blue, and the color intensity can be measured with a spectrophotometer. The TLR reporter cell lines (InvivoGen) were cultured in DMEM containing 10% FBS and 1× antibiotics. When the cells reached 80% confluent, they were washed with PBS, and detection medium was added to harvest the cells using a scraper. Cells (5 × 10⁴ cells/well; 180 μl) were seeded in a 96-well plate, and 20 μl of PBS, samples, PAM3 (Invitrogen, Waltham, Massachusetts, USA), LPS (Invitrogen), poly I:C (Invitrogen), flagellin (Invitrogen), R848 (Invitrogen), or CpG-ODN (Invitrogen) was added to each well. Then, the cells were cultured at 37 °C, 5% CO₂, and 90% humidity for 20 h, and the absorbance at 630 nm was recorded.