P-FPMI-95 - P-FPMI-95
LPS was isolated from P. aeruginosa H103 using the Darveau-Hancock method as previously described*. Briefly, P. aeruginosa was grown overnight in LB broth at 37ºC. Cells were collected, washed and the isolated LPS pellets were back extracted with a 2:1 chloroform:methanol solution to remove contaminating lipids. Purified LPS were further quantitated using an assay for 2-keto-3-deoxyoctosonic acid (KDO assay) and then resuspended in endotoxin-free water (Sigma-Aldrich). The cationic peptide, human LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFFRNLVPRTES), was synthesized using F-moc chemistry at the Nucleic Acid/Protein Synthesis Unit, University of British Columbia (Vancouver, BC, Canada). The synthetic peptide was re-suspended in endotoxin-free water and stored at -20 degree Celcius until further use. Primed THP-1 cells were stimulated with LPS (100 ng/ml) in the presence of LL-37 (20 ug/ml) for 4 hours.
* Darveau, RP and Hancock, REW. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 155, 831-8 (1983).