Dendritic cell pellets (1–6 million cells) were lysed in non-ionic detergents (4% CHAPS and 4% Triton X-100) in the presence of protease inhibitors (EDTA-free, Roche) and 590 units of nuclease (US Biologicals) for 45 min at 4°C with rotation. The cell lysate was clarified by centrifugation at 112,000 g for 30 min at 4°C.
Immuno-isolation of MHC II complexes. An isotype IgG (Southern Biotech) and the pan anti-MHC II class monoclonal antibody (L243) (BioXCell) were each coupled to individual HiTrap NHS-activated HP columns (GE Healthcare). The two columns were connected in series with the Isotype IgG column first for the immuno-isolation process. The cleared lysate was loaded on the immuno-isolation columns. The Isotype IgG column was removed, and the MHC II complexes were washed with a buffer and then eluted from the L243 column with 10% acetic acid. The MHC II peptides were desalted by solid-phase extraction using an MCX plate (Waters) into LoBind 96 well plates (Eppendorf) and then transferred to MS plates (Abgene), and vacuum evaporated.
Immuno-isolation of MHC II complexes. An isotype IgG (Southern Biotech) and the pan anti-MHC II class monoclonal antibody (L243) (BioXCell) were each coupled to individual HiTrap NHS-activated HP columns (GE Healthcare). The two columns were connected in series with the Isotype IgG column first for the immuno-isolation process. The cleared lysate was loaded on the immuno-isolation columns. The Isotype IgG column was removed, and the MHC II complexes were washed with a buffer and then eluted from the L243 column with 10% acetic acid. The MHC II peptides were desalted by solid-phase extraction using an MCX plate (Waters) into LoBind 96 well plates (Eppendorf) and then transferred to MS plates (Abgene), and vacuum evaporated.