4.5. Reporter Assay for TLR Receptor Activation

TLR2 and TLR9 binding by corynebacteria was analyzed by infection of HEK Blue 293 hTLR2 and hTLR9 cells (InvivoGen, San Diego, CA, USA). HEK Blue 293 cells carry a stably integrated NFкB- and AP1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter construct. The assay was carried out according to a protocol published by Weerasekera and co-workers [34]. Cells were seeded in 96-well plates, infected with viable and UV-killed bacteria at MOI values of 100, 10 and 1 and incubated at 37 °C in a CO₂ incubator. Heat-killed L. monocytogenes (HKLM) were used as positive control and PBS buffer as negative control. After an incubation period of 16 h, the 96-well plates were centrifuged (350× g, 5 min) and 20 µL of the cell-free supernatant was mixed with 180 µL pre-warmed SEAP detection reagent QUANTI-Blue (InvivoGen, San Diego, CA, USA) according to the manufacturer’s protocol. After a 2 h incubation under cell culture conditions, the absorbance at 620 nm was measured using a microplate reader (TECAN Infinite M nano plus, Männerdorf, Switzerland). HEK Blue 293 hTLR9 cells were infected with MOI 1 and MOI 50 with viable and UV-killed bacteria in HEK Blue detection medium and incubated for 24 h under cell culture conditions. SEAP activity was measured as mentioned above.