Quantifying TLR5-Mediated CXCL8 Response

The read-out for TLR5 activity in all experiments was CXCL8 secretion into cell line culture supernatants. Cell lines were prepared at 5 × 10⁵ cell per ml and 180 μl/well was added to each well in a 96-well plate, with the addition of 20 μl/well flagellin at final concentrations as required (see results). Following overnight incubation, cell culture supernatants were harvested for assays. Human CXCL8 protein levels were measured by ELISA using the BD OptEIA human IL-8 ELISA set (BD Biosciences) following the manufacturer’s instructions. Bovine CXCL8 protein release was measured by ELISA as described previously^{7 (link)}. All ELISAs were carried out using Nunc-Immuno Maxisorp 96 well plates, with all samples and standards in triplicate. The plates were read using a Synergy HT plate reader (BioTek).