Multiple B-progenitor cell subsets are represented in HB-LTC. Nonadherent cells from HB-LTC were collected at 6 to 8 weeks for FACS analysis. (A) Two-color analysis demonstrating the presence of HB-LTC CD19⁺ progenitors expressing surrogate light chain (VpreB), cytoplasmic μ (Cμ), or surface IgM. (B) Analysis demonstrating independent expression κ versus λ light chains and of VpreB versus κ or λ light chain. (C) Analysis demonstrating coexpression of IgM and IgD within HB-LTC immature IgM⁺B-cell population. (D) Example of three-color analysis of purified HB-LTC CD19⁺ progenitors showing staining of VpreB versus cμ. This analysis showed VpreB⁻cμ⁻IgM⁻ (R1) progenitors; VpreB⁺cμ⁻IgM⁻(R2) and VpreB⁺cμ⁺IgM⁻ (R3) subsets; and VpreB⁻cμ⁺IgM⁻and VpreB⁻cμ⁺IgM⁺ subsets (R4; IgM data not shown). Results are representative from more than eight independent experiments. Numbers indicate the percentage of cells in each quadrant.