Fig. 1.

In vitro generation of BCR^neg B cells. (A) Schematic showing Cre-mediated deletion of the loxP-flanked B1-8 allele and subsequent loss of BCR expression. (B) Splenic B cells from B1-8f/+, bcl-2tg mice were treated with recombinant TAT-Cre for 45 min, cultured for 9 d, and sorted by FACS to >98% purity based on IgM expression (data are representative of two biological replicates). Serial dilutions of whole-cell lysates starting at 300,000 cell equivalents were analyzed for Igμ and β-actin expression by Western blotting. (C) Splenic B cells from B1-8f/+, bcl-2tg mice were transduced with TAT-Cre for 45 min, cultured for 5 d in medium, and stained for CD19, IgM, and the indicated surface markers (data are representative of three biological replicates). BCR^pos cells (open histograms) and BCR^neg cells (closed histograms) were gated as shown.