CX3CR1⁺ and CX3CR1⁻ ETPs have comparable DC potential in vitro. (A) BM-derived progenitors (CLPs, pre-DCs, CDPs, MDPs, and MPs) and thymic progenitors (CX3CR1⁺ and CX3CR1⁻ ETPs) were sorted from CX3CR1^GFP/+ reporter mice. Progenitors were sorted according to gates shown in Figures 1 and 2. CLPs were defined as lin⁻Sca-1⁺CD117^intCD127⁺ CD135⁺. Purified precursors were cultured on OP9 stromal cells in the presence of Flt-3L, SCF, IL-7, and GM-CSF, and analyzed at days 4 and 6 to assess their myeloid and B-lineage potential. Cells derived from these precursors were defined as DC, based on expression of CD11c, along with Sirpα and B220. Bona fide macrophages were identified as CD11b⁺CD11c⁻, while B220 cells were defined as CD11c⁻CD11b⁻B220⁺CX3CR1⁻. (B) Quantification of data shown in (A) at day 4 (upper panel) and day 6 (bottom panel) of culture. Numbers above the columns indicate fold expansion of precursors compared with day 0. (C) Quantification of DC populations shown in (A, middle panel) at day 4 (upper panel) and day 6 (bottom panel) of culture. Development of 3 major DC populations was assessed: CD11c⁺Sirpα⁺, CD11c⁺Sirpα⁻, and CD11c⁺B220⁺ (pDC). (D) Expansion of precursors between days 4 to 6 inversely correlated to the frequency of myeloid cells generated by day 6. (E) The potential of CX3CR1⁺ and CX3CR1⁻ ETPs to develop toward the myeloid or B lineages was assessed by limiting dilution assay on OP9 stromal cells in the presence of Flt-3L, SCF, IL-7, and GM-CSF. (A-E) Data are representative of 2 independent experiments.