50 kda mwco dialysis tubing

DOTAP-Nano was prepared with the NanoAssemblr Benchtop (Precision NanoSystems Inc., BC, Canada), which can mediate bottom-up self-assembly for nanoparticle synthesis with microfluidic mixing technology. Briefly, DOTAP was dissolved in ethanol. The lipid solution (10 mg/mL) in ethanol and 25 mM acetate buffer (pH 4.0) were injected into the microfluidic mixer at a 1:3 volume and at a combined final flow rate of 15 mL/min (3.75 mL/min ethanol, 11.25 mL/min aqueous). The DOTAP-Nano mixtures were immediately dialyzed (50 kDa MWCO dialysis tubing; Repligen Corporation, MA) against 5% glucose solution to remove ethanol. DOTAP-Nano was then concentrated to approximately 1.5–2.0 mg/mL DOTAP by using Amicon Ultra Centrifugal filters (100 kDa MWCO; Merck KGaA, Darmstadt, Germany) and sterilized through a 0.22 μm PVDF filter (Merck KGaA). The preparation of all lipid nanoparticles was performed at room temperature.

CpG containing adjuvant D35/DOTAP was prepared with NanoAssemblr Benchtop (Precision NanoSystems Inc., BC, Canada), which can mediate bottom-up self-assembly for nanoparticle synthesis with microfluidic mixing technology. D35 (an A-type CpG ODN) [33 (link), 34 (link)] was dissolved at 500 μg/mL in 25 mM sodium acetate at pH 4.0. One volume of D35 solution and three volumes of 10 mg/mL DOTAP in ethanol were injected into the microfluidic mixer with a combined final flow rate of 15 mL/min (3.75 mL/min ethanol, 11.25 mL/min aqueous). The D35/DOTAP mixtures were immediately dialysed (50 kDa MWCO dialysis tubing; Repligen Corporation, MA) against 5% glucose solution to remove ethanol and unload D35. D35/DOTAP was filtered through a 0.22-μm PVDF filter (Merck KGaA).
For protein immunisation, 10 μg of the gH/gL/gQ1/gQ2 tetrameric complex and 10 μg of D35/DOTAP (containing D35 amount) were mixed and then made up to a volume of 100 μL with phosphate-buffered saline (PBS).