Pyrogene recombinant factor c kit

Animals were fed ad libitum on either a high protein, moderately low carbohydrate, moderately low-fat diet (P60, C20, F20; henceforth, HP); a low protein, high carbohydrate, moderately low-fat diet (P5, C75, F20; henceforth, HC), or a low protein, moderately low carbohydrate, high-fat diet (P5, C20, F75; henceforth, HF). These three diets represented the extremes (apices) from a larger set of 10 diet compositions (Supplementary Table 1) that were used in a subset of the experiments, encompassing a macronutrient range of protein (5–60%), carbohydrate (20–75%), and fat (20–75%) chosen using nutritional geometry to comprehensively sample dietary macronutrient mixture space^{39 (link)}. All diets were isocaloric at 14.5 MJ/kg and based on a modification to the semi-purified AIN93G formulation and purchased from Specialty Feeds, Glenn Forest, Australia (SF17-188-SF17-197) and the HP/HC/HF used for most experiments had very low endotoxin levels as tested with the PyroGene Recombinant Factor C kit (Lonza).

HEK293 clones expressing human TLR2, TLR9 and S. aureus LTA (LTA-SA) were purchased from InvivoGen (San Diego, California, USA). Human Hb, blasticidin, and hygromycin were from Sigma-Aldrich (St. Louis, MO, USA). Buffy coat from healthy donors was obtained from the Blood Bank, National University Hospital, Singapore. Primary blood cells were incubated in 5% CO₂ at 37 °C in HEPES-buffered RPMI 1640 containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 2% FBS. HEK293 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Adult Human Hb, which contains 96.5–98.5% HbA1 (α₂β₂ dimer) and 1.5–3.5% HbA2 (α₂δ₂ dimer), has been verified to be in the metHb state by spectrophotometric scanning between 500 and 700 nm (Du et al., 2010 (link)). As the endotoxin concentration in Hb was determined to be 2.86 EU/mg/ml by PyroGene Recombinant Factor C kit (Lonza Inc.), 1 mg/ml of Hb was pre-treated with 5 μg/ml of polymyxin B, which can scavenge 10 EU/ml of endotoxin. The metHb + LTA complex was pre-formed by co-incubating 1 mg/ml metHb with 10 μg/ml LTA for 30 min.