Three-color immunofluorescence analysis of early B-lineage cells in BM based on intracellular VpreB expression.

BM cells from 3 controls of different ages, 2 XLA patients (P3 and P12), and 1 B-cell⁻ SCID patient were fixed, permeabilized, and then stained with PE-labeled anti-VpreB and PC5-labeled anti-CD19 mAbs in combination with FITC-labeled anti-TdT mAb or antihuman μH chain Ab. The corresponding fluorochrome-labeled irrelevant mAbs or Abs were used as controls. The cells were gated for lymphoid cells by light scatter characteristics upon a flow cytometer and analyzed in the 3-color manner. (A) As demarcated in the figure, the combined use of anti-VpreB and anti-CD19 mAbs appeared to detect the presumably whole populations of B-lineage cells in BM. (B) The relation between VpreB and the μH chain or TdT expressed by BM-lineage cells, which were gated by a combined use of CD19 and VpreB expression. The number indicates the percentage of cells in each quadrant.