BLyS binding and BLyS requirement of ABCs. (A) Surface expression of BLyS receptors and BLyS binding capacity of ABCs (dark bold) compared with FO (dotted) and MZ (dashed) B cells from aged mice. (B) Flow cytometrically sorted splenic ABC and FO B cells from young and aged mice were cultured separately in the absence (medium only) or presence of BLyS. Living cells were TO-PRO-3 negative. (C) Effect of BLyS neutralization in vivo. Mice were treated with 100 μg of anti-BLyS or isotype control IgG1 IP on days 0 and 5. Numbers of FO, MZ and ABCs are shown at 21 days after the first injection. Mean with SEM are shown; each symbol represents one mouse. Similar results were observed from 3 independent experiments.