In vivo Uptake of Fluorescent Antigens

In vivo uptake studies were carried out following procedures we previously developed (60 (link)): BALB/c mice were immunized with NE formulations containing 10 μg of AF647-eOD-GT8, 5 μg of green fluorescent BODIPY FL C₁₂ cholesteryl ester, 5 μg of 3M-052, and 0.1 μg of squalene oil. Twenty-four hours later, mesLNs were collected and single-cell suspensions were prepared. Cells were first incubated with live/dead stain (Zombie UV, BioLegend) at 1:750 dilution in 100 μl of PBS for 15 min at 25°C and then with antimouse CD16/32 antibody (TruStain FcX, BioLegend) at 1:100 dilution in 50 μl of flow cytometry buffer for 10 min at 25°C. Cells were stained against antimouse CD3ε PE-CF594 (clone 145-2C11; BD Biosciences), CD19 BUV395 (1D3; BD Biosciences), CD11b BUV737 (M1/70; BD Biosciences), CD11c BV711 (N418, BioLegend), F4/80 PE (BM8; BioLegend), Ly6C BV785 (HK1.4, BioLegend), and Ly6G BV421 (1A8, BioLegend) at 1:100 dilution in 100 μl of flow cytometry buffer for 15 min at 25°C. Cells were washed twice and fixed using 2% PFA. Data acquisition was performed using BD FACSymphony A3 and analyzed with FlowJo 10.