Identification of memory anti-Gal B cells by binding of FITC–α-gal–BSA. Flow cytometry of spleen lymphocytes identified as anti-Gal B cells by double staining with FITC–α-gal–BSA and PE-antimouse Ig. Staining of lymphocytes in control mice (ie, KO mice receiving 20 × 106 lymphocytes that include memory anti-Gal B cells) is shown in left panel; staining in mice that received the same lymphocytes but that were tolerized by WT lymphocytes is shown in right panel. Both groups were immunized with PKM on days 14 and 21 and staining of lymphocytes was performed on day 28 after adoptive transfer. Note that approximately 1% of B cells in control recipients of memory anti-Gal B cells bind FITC–α-gal–BSA, whereas no such cells are detected in the tolerized mice. Data are from 1 representative mouse out of 4 per group.
