Mannan and polyinosinic–polycytidylic acid sodium salt were obtained from Sigma-Aldrich (St. Louis, Mo). Lipoteichoic acid was obtained from Sigma-Aldrich (St. Louis, Mo) and InvivoGen (San Diego, CA). Resiquimod was obtained from Tocris Bioscience (Minneapolis MN). Anti-mouse CD40 (clone: FGK4.5/GFK45) was obtained from BioXCell (West Lebanon, NH). Biocompatible anchor for cell membrane (BAM) was obtained from NOF America Corporation (White Plains, NY).
Mannan-BAM synthesis was performed as previously reported.[9 (link),11 (link),14 (link)] Aminated Mannan was prepared by reductive amination.[42 (link)] Mannan solution in an environment of ammonium acetate (300 mg ml−1) was reduced by 0.2 M sodium cyanoborohydride at pH 7.5 and 50 °C for five days. Solution was further dialyzed using MWCO 3500 dialysis tubing (Serva, Heidelberg, Germany) against PBS at 4 °C overnight. Binding of BAM on amino group of Mannan was performed at pH 7.3 according to Kato et al.[10 (link)] During one hour at room temperature N-hydroxysuccinimide (NHS) group of BAM reacted with amino group of Mannan. Solutions obtained after dialysis as above was stored frozen at −20 0C until use.
Mannan-BAM synthesis was performed as previously reported.[9 (link),11 (link),14 (link)] Aminated Mannan was prepared by reductive amination.[42 (link)] Mannan solution in an environment of ammonium acetate (300 mg ml−1) was reduced by 0.2 M sodium cyanoborohydride at pH 7.5 and 50 °C for five days. Solution was further dialyzed using MWCO 3500 dialysis tubing (Serva, Heidelberg, Germany) against PBS at 4 °C overnight. Binding of BAM on amino group of Mannan was performed at pH 7.3 according to Kato et al.[10 (link)] During one hour at room temperature N-hydroxysuccinimide (NHS) group of BAM reacted with amino group of Mannan. Solutions obtained after dialysis as above was stored frozen at −20 0C until use.
