Figure 4

Isolation of rabbit antigen-specific plasma/plasmablast cells (ASPCs) using fluorescence-activated cell sorting (FACS). (A) A representative FACS graph of the lymphocytes of green fluorescent protein (GFP)-immunized rabbits stained with anti-rabbit IgG Dylight 650, ER-tracker and GFP Dylight 488. The forward-versus-side-scatter (FSC vs SSC) with gate R1 represents lymphocytes. Plasma/plasmablast cells (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) R1-gated, R2-gated, R3-gated and R4-gated cells stained intracellularly with anti-rabbit IgG Dylight 594 (red) and GFP-Dylight 488 (green). The numbers indicate the mean percentages of cells exhibiting IgG and GFP signals from two separate experiments. (C) A representative agarose gel electrophoresis of cognate pairs of V genes amplified from single-cell-sorted R3-gated and R4-gated cells (upper). Polymerase chain reaction (PCR) success ratio for V genes from single-cell-sorted R3-gated and R4-gated cells (lower). (D) The antigen specificity of rabbit monoclonal antibodies (mAbs) produced from R3-gated and R4-gated cells. Cognate pairs of linear IgH and IgL genes were cotransfected into 293FT cells, and the concentration of antibodies in the cell culture supernatant was determined (upper). The antigen specificity of the mAbs was expressed as relative light units (RLU)/IgG (lower).