HEK 293 cells stably transfected with the murine TLR5 (InvivoGen, Toulouse, France) were cultured in DMEM (Biochrome, Berlin, Germany) containing 10% FCS (Biochrom, Berlin, Germany), L-glutamine (0.15 mg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and blasticidin (10 µg/ml) (InvivoGen). Wild-type HEK 293 cells were cultured without blasticidin as indicated above. For TLR5-activation assays, 4 × 104 cells per well were seeded in 48-well plates and cultured in DMEM containing 2% FCS, L-glutamine, penicillin, and streptomycin overnight. Cells were stimulated for 22 h with equimolar amounts rFlaA, rFlaA:Artv1, or rFlaA:Artv1hyp. Subsequently, human IL-8 secreted in the supernatant was quantified by ELISA using the BD OptEIA human IL-8 ELISA Set (BD Biosciences, Heidelberg, Germany).
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