Rdsg3 antigen

Dsg-specific memory B cells were measured using a polyclonal stimulation assay, essentially as previously described (Pinna et al., 2009 (link)). Briefly, PBMCs were cultured at 1×10⁶ cells/mL in RPMI supplemented with penicillin/streptomycin, L-glutamine, 10% FBS, a 50 μM of beta-mercapto-ethanol (Sigma) (R10) containing R848 (1 μg/mL, Invivogen) and recombinant human-IL2 (10 ng/mL, Biolegend) for 3 days at 37°C. Total and Dsg3-specific IgG-secreting cells were quantified by ELISPOT assay, in which a 96-well ELISPOT filter plates (Millipore) were coated overnight with rDsg3 antigen (3 μg/mL, provided by the Hertl Lab), rDsg1 antigen (3 μg/mL, EuroImmun), or polyvalent goat anti-human Ig (25 μg/mL, Rockland) in PBS/1 mM CaCl₂. Plates were washed and blocked by incubation with R10 at 37°C for 2 h. PBMCs were added to the plates in a dilution series and incubated for 5 hs at 37°C. Plates were washed with PBS, followed by PBS/0.05% Tween, and incubated with biotinylated anti-human IgG antibody (Invitrogen) at room temperature for 90 min. After washing, plates were incubated with an avidin-D horseradish peroxidase conjugate (Vector laboratories) and developed using 3-amino-9-ethyl-carbazole substrate (Sigma). Plates were scanned and analyzed using an automated ELISPOT counter (CTL, Cellular Technologies).