A novel culture method (iCD103) preferentially and efficiently generates CD103^hi CD8α-like DCs. BM cells were cultured to generate GM-DCs, FL-DCs, or iCD103-DCs. (A) iCD103-DCs were analyzed by flow cytometry. Histograms show CD8α-like DC marker expression (solid black line) of live-gated CD11c⁺B220^– DCs compared with isotype controls (gray overlay). (B) Clec9A vs CD103 (left) and Clec9A vs SIRPα (right) expression of live-gated CD11c⁺B220^– iCD103-DCs. (C) Yield comparison per 1 × 10⁶ input BM cells of CD11c⁺B220^– DCs (left) and CD103⁺CD11c⁺B220^– DCs (right) using GM-DC, FL-DC, and iCD103-DC culture methods. (D) Typical light micrographs of GM-DC and iCD103-DC cultures during steady state (Medium, upper panel) or after overnight CpG stimulation (Activated, lower panel). Enlarged views of activated DCs are depicted. Scale bars represent 50 µm. (E) Bright-field micrographs of cytospins of sorted CD11c⁺ GM-DCs and CD11c⁺B220^–CD103⁺ iCD103-DCs after Giemsa staining. (F) Fluorescence micrographs of CpG-activated GM-DCs and iCD103-DCs stained for actin (magenta), CD103 (yellow), and nuclei (DAPI; cyan). (E-F) Scale bars represent 10 µm. (G) Myeloid lineage marker expression among live-gated CD11c⁺ GM-DCs or CD11c⁺B220^– cDCs (FL-cDCs and iCD103-cDCs). Specific (solid black lines) and isotype control stainings (gray overlay) are depicted. Results are representative of 3 to 4 (A-B) or 3 (D-G) independent experiments. Data for (C) were combined from 7 (GM-DC), 3 (FL-DC), and 6 (iCD103-DC) independent experiments, each involving cultures from 1 to 6 mice. **P < .01, ***P < .001; 2-tailed Mann-Whitney U test.