B-cell depletion in old 3-83Tg mice revives B lymphopoiesis in the BM and rejuvenates the peripheral repertoire. Old B10D2 3-83Tg mice (20 months, confirmed to have an old-like B-cell phenotype by blood-sample staining) were treated for B-cell depletion. This was achieved by an initial intraperitoneal injection with a mixture of monoclonal antibodies specific to B220 (150 μg/mouse), CD19 (150 μg/mouse), and CD22 (150 μg/mouse), followed by a second intraperitoneal injection of rat anti–mouse κ monoclonal antibodies (150 μg/mouse) 48 hours later. Mice were bled 3 days after last injection to ensure more than 90% B-cell depletion in peripheral blood. After 65 days, the BM and spleen were analyzed for the B lineage by flow cytometry. (A) Peripheral blood staining of representative young and old 3-83Tg mice. Old mice were prescreened to have less than 50% B cells in peripheral blood expressing the transgenic receptor. (B) Absolute numbers of B220⁺ cells were calculated based on the total number of nucleated cells purified from the BM (2 femurs and 2 tibias) or spleen (n = 4 mice in each group). *Significant difference (P < .05). (C) BM cells were analyzed by flow cytometry with a lymphocyte gate as defined by light scatter. Expression of the transgenic receptor was detected using an anti–3-83 clonotype specific monoclonal antibody 54.1. (D) Spleen cells from the indicated mice were analyzed by flow cytometry. Analysis for B220 and PanCD45 was conducted on gated CD19⁺ cells. The plots shown are representative of 4 mice in each group.