Figure 5

Isolation of rat antigen-specific plasma/plasmablast cells (ASPCs) using fluorescence-activated cell sorting (FACS). (A) A representative FACS graph of the lymphocytes from green fluorescent protein (GFP)-immunized rats stained with anti-rat IgG Dylight 650, ER-tracker and GFP-Dylight 488. The forward-versus-side-scatter (FSC vs SSC) with gate R1 represents lymphocytes. Plasma/plasmablast cell (PCs) were gated as IgG^low endoplasmic reticulum (ER)^high (R2). The R2-gated cells were further subdivided into the ASPCs (IgG^low ER^high GFP⁺, R3 gate) and non-specific PCs (IgG^low ER^high GFP^-, R4 gate). The numbers indicate the mean percentages of cells in the gated area from three separate experiments. (B) The R3-gated and R4-gated cells stained intracellularly with anti-rat IgG Dylight 594 (red) and GFP Dylight 488 (green). The numbers indicate the mean percentages of cells with IgG and GFP signals from three separate experiments. (C) Representative agarose gel electrophoresis of cognate pairs of V genes amplified from single-cell-sorted R3-gated and R4-gated cells. (D) Antigen specificity of the rat monoclonal antibodies (mAbs) produced from R3-gated and R4-gated cells. Cognate pairs of linear IgH and IgL genes were cotransfected into 293FT cells. The antigen specificity of the mAbs was expressed as relative light units (RLU)/IgG.