Bone marrow-derived macrophages were obtained from tibias and femurs of mice as per protocols PP0000004702 (Harvesting and Plating Thioglycollate Elicited Macrophages for LM Distribution, pdf) and PP0000004602 (ATP / Kdo2-Lipid A treatment, pdf). Cell treatments consisted of a) Control (no treatment); b) Kdo2-Lipid A pretreatment only (4hr pretreamemt, no ATP); c) ATP only (with no Kdo2-Lipid A pretreatment); d) Kdo2-Lipid A pretreatment (4 hrs), followed by addition of ATP at t=0. Lipid/mRNA measurements were taken at t=0 (control only), 15m, 30m, 1h, 2h, 4h, 8h, and 20h (relative to ATP addition at t=0). Measurements were normalized to the average DNA value across all timepoints for each treatment and expressed as pmol analyte/ug DNA.
Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.
Raetz C, Garrett T, Reynolds C, Shaw W, Moore J, Smith JD, Ribeiro A, Murphy R, Ulevitch R, Fearns C, Reichart D, Glass C, Benner C, Subramaniam S, Harkewicz R, Bowers-Gentry R, Buczynski M, Cooper J, Deems R and Dennis E. Journal of Lipid Research 47, 1097-1111 (2006)