Memory B cell assay

Antigen-specific memory B cells (MBC) were detected essentially as previously described [30]. Briefly, PBMC were cultured at 1x10⁶ cells per ml of R10 supplemented with 50 uM B-mercaptoethanol (Sigma-Aldrich) and polyclonally stimulated with pokeweed mitogen extract (1 μg/mL, Sigma-Aldrich), phosphothiolated CpG ODN-2006 (6 μg/mL, Invivogen), and Staphylococcus aureus Cowan (1:10,000, Sigma-Aldrich) for 6 days. After in vitro stimulation, total and V. cholerae specific IgM, IgG, and IgA cells were quantified by ELISPOT assay, as described above.