Fig. 4.

Detection and isolation of Ag-specific IgG1/2b⁺B cells from immunized human immunoglobulin transgenic rats. After immunomagnetic enrichment with Ag tetramers and staining with fluorescent mAbs, cells were analyzed on flow cell sorter. a Dots plots show viable and singlets lymphocytes stained with anti-IgG1/2b and anti-TCR (clone R7.3). MAbs from one unimmunized and one immunized human immunoglobulin transgenic rats is shown. b Dots plots show B cells stained with relevant Ag tetramers on unimmunized (left) and immunized (right) human immunoglobulin transgenic with β-Gal, CD22 and HLA-A2/Pp65 Ags. Cells were also stained with irrelevant Ag tetramers to exclude unspecific B cells. Cells from human immunoglobulin transgenic rats immunized with β-Gal and CD22 were stained with ovalbumin tetramers to exclude streptavidin specific B cells. Cells from human immunoglobulin transgenic rats immunized with HLA-A2/Pp65 were stained with HLA-B7/irrelevant peptide tetramers to exclude streptavidin and pan class I MHC-specific B cells, or with HLA-A2/irrelevant peptide tetramers to exclude streptavidin and HLA-A2 specific B cells