Opposite responses of T1 immature and mature B cells to BCR triggering. (A) Fluorescence activated cell sorting (FACS) of magnetically separated, untouched B lymphocytes from the spleens of C57BL/6 mice using APC-conjugated CD93-specific and PE-conjugated CD23-specific antibodies. T1 immature (CD93+, CD23-) and mature (CD93-, CD23+) B cells were sorted, as shown by the two rectangles. (B-E) Proliferation and apoptosis of sorted mature (M) and T1 immature (IM) B cells in response to BCR stimulation. Cells were incubated in vitro with (BCR) or without (ctrl) anti-μ F(ab')2 for the indicated periods of time and assayed for (B) ATP content, (C) caspase 3/7 activity and (D) trypan blue (TB) exclusion. Values are mean ± s.e.m. of three independent experiments. *, P < 0.01, compared to the respective control samples using Student's t-test (black and grey asterisks in (D) refer to the comparison of mature and immature samples, respectively). RFU, relative fluorescence units. (E) Phase contrast microscopy of mature and T1 immature B cells after 48 h of stimulation with anti-μF(ab')2 in culture. Lower panels show BCR-triggered large clusters of proliferating mature cells (left) and smaller groups of mainly apoptotic T1 immature B cells (right). Scale bar, 50 μm. Non-B cells from spleen did not respond to the treatment with anti-μ IgM antibodies, as determined by these assays (data not shown).