Intranasal Administration of DQ-OVA

Fifty microliters of PBS, DQ-OVA (50 μg), and flagellin (3 μg total) adjuvanted DQ-OVA (50 μg) were applied via the nostrils as recently described (28). DQ-OVA was used for degradation and accumulation assays as it consists of OVA bound to a self-quenching fluorescent dye, which upon intracellular degradation releases specific fluorescence (excitation at 505 nm, emission at 515 nm). Accumulated DQ-OVA forming dimers emit fluorescence in a different channel (excitation at 488 nm, emission at 613 nm). Animals were euthanized 4 or 24 h after intranasal administration and lung and or lung draining lymph nodes were harvested to determine the uptake, trafficking, phenotype, and antigen degradation (28). Tracking the quantity and localization of specific APC in vivo was achieved via the use of a SIINFEKL monoclonal Ab (mAb), which specifically reacts with ovalbumin-derived peptide SIINFEKL bound to H-2Kb of major histocompatibility complex (MHC) class I. To enumerate the antigen cross presentation, DCs from the draining lymph nodes were stained with PE anti-mouse MHC class I molecule (Kb) bound to the peptide SIINFEKL Abs (Kb-SIINFEKL) (BioLegend; San Diego, CA). Data was acquired on BD Fortessa flow cytometer.