TLR5-activation assay

SS Stefan Schülke
KK Kirsten Kuttich
SW Sonja Wolfheimer
ND Nadine Duschek
AW Andrea Wangorsch
AR Andreas Reuter
PB Peter Briza
IP Isabel Pablos
GG Gabriele Gadermaier
FF Fatima Ferreira
SV Stefan Vieths
MT Masako Toda
SS Stephan Scheurer
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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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