Characterizing Antigen-Specific B Cells in Mice

GC responses were analyzed as previously reported (60 (link)): BALB/c mice were immunized with NE formulations containing 10 μg of eOD-GT8, 5 μg of 3M-052, and 0.1 μg of squalene oil. After 12 days, 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 10 min at 25°C and then with antimouse CD16/32 antibody at 1:100 dilution in 100 μl of fluorescence-activated cell sorting buffer for 10 min at 25°C. Cells were stained against antimouse CD90.2 BV785 at 1:200 (clone 30-H12; BioLegend), CD19 BUV395 at 1:200 (1D3; BD Biosciences), CD38 FITC at 1:200 (90; BioLegend), GL7 PerCP-Cy5.5 at 1:150 (GL7; BioLegend), IgA AF647 at 1:100 (SouthernBiotech), eOD-tetramer BV605 at 1:100, and eOD-tetramer BV421 at 1:100 [tetramers formed by incubating 4 equivalents of biotinylated eOD with 1 equivalent of streptavidin-BV605 or streptavidin-BV421 (BioLegend) 18 hours at 4°C, prior to staining] in 100 μl of flow cytometry buffer for 20 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.