Dppc 1 2 dipalmitoyl sn glycero 3 phsophocholine

SMNP adjuvant was prepared as described (77 ). Cholesterol (20 mg/ml; Avanti Polar Lipids, catalog no. 700000) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phsophocholine: 20 mg/ml; Avanti Polar Lipids, catalog no. 850355) were dissolved in Milli-Q water containing 20% w/v MEGA-10 (Sigma-Aldrich, D6277) detergent at 60°C. Monophosphoryl lipid A (10 mg/ml; Avanti Polar Lipids, catalog no. 699800P) was dissolved in 20% w/v MEGA-10 at 37°C. Quil-A saponin (InvivoGen; vac-quil) was dissolved in Milli-Q water at a final concentration of 100 mg/ml at 37°C. All components were then mixed at a mass ratio of 10:10:2.5:1 [Quil-A:cholesterol:DPPC:MPLA (Monophosphoryl lipid A)] at 60°C followed by dilution with phosphate-buffered saline (PBS) to a final concentration of cholesterol (1 mg/ml). The solution was allowed to equilibrate over night at room temperature, followed by dialysis against PBS using a 10k molecular weight cutoff (MWCO) membrane. The adjuvant solution was then sterile filtered, concentrated using 50k MWCO centricon spin filters, and further purified by fast protein liquid chromatography using a Sephacryl S-500 HR size exclusion column. Cited concentrations of SMNP refer to the concentration of saponin injected.