Adult spleen contains progenitors of LTC-DCs. (A) Adult splenocytes depleted of T and B cells were sorted into populations of c-kit⁻CD11c⁻ and c-kit⁺CD11c⁻ cells and cocultured over STX3 to evaluate potential for DC development. (B) Nonadherent progeny cells collected from cocultures at 13, 23, and 30 days were assessed for DC production flow cytometrically. Cells were stained simultaneously for CD11c, CD11b, CD8α, and MHC-II. FSC and SSC parameters were used to distinguish large cells for multichannel marker analysis. Isotype controls were used to define background antibody binding (zebra plots) and set gates. The percentage of cells showing specific binding is indicated within gates. Staining for CD8 was negative (data not shown). (C) DC production from 13-, 23-, and 30-day cocultures established with adult c-kit⁻CD11c⁻ and c-kit⁺CD11c⁻ spleen cell subsets is summarized. Cells produced were identified as either CD11c^loCD11b^hiMHC-II⁻ (MHC-II⁻ LTC-DC) cells or CD11c^hiCD11b^loMHC-II^lo/+ (MHC-II⁺ DC) cells. Cell production is shown in terms of absolute number and yield of DCs, or percentage DCs among total cells produced in cocultures.