Yield of hBcl-2 Transgenic B Cells

Transgenic mice expressing human bcl-2 have enlarged spleens and demonstrate a polyclonal increase in the number of B cells in the spleen, lymph nodes, and bone marrow (5-7). We found that hbcl-2 transgenic mouse spleens (146 ± 19 mg, n = 14) were, on average, twice as large as spleens from wild-type littermate mice (76 ± 11 mg, n = 7) and contained four times as many B cells (Table 1). hBcl-2 transgenic B cells could be enriched to >95% by depletion of cells expressing CD3 (T cells), CD11b (myeloid cells, i.e., macrophages and neutrophils), and CD43 (expressed on most leukocytes, but not resting mature B cells) using the Miltenyi AutoMACS magnetic cell sorter (AfCS Procedure PP0000000100). However, this method nonspecifically removed some B cells, possibly due to B cells adhering to T cells or myeloid cells, or by B cell Fc receptor (FcR) interactions with antibody-coupled magnetic beads. Since the cell yields we obtained were sufficient (60%), and we wished to compare wild-type and bcl-2 transgenic B cells prepared with the same procedure, we did not further optimize the isolation protocol specifically for hbcl-2 transgenic B cells, other than to take total cell numbers per spleen into account for calculating the amounts of beads required for labeling, and for the processing of the bead-labeled cells.