The splenic cDC compartment matures asynchronously during early post-natal development. (A) Representative flow-cytometric analysis for the identification and quantification of the different cDC subsets. cDCs are defined as CD64⁻F4/80^lowLineage(CD3e/CD19/NK1.1/Ter119)⁻MHC-II⁺CD11c⁺CD26⁺ cells. (B) Quantification of WT splenic cDC subsets during early post-natal development. The number and frequency of the different subsets are both shown. The rate of cDC growth is calculated from the first 28 d of post-natal development. n = 9, day 2 samples; 9, day 7 samples; 7, day 14 samples; 7, day 21 samples; 6, day 28 samples; and 9 adult samples; data verified in at least two different experiments per time point; two-way ANOVA with Sidak’s multiple comparisons post-test. (C) Representative flow cytometry plots and quantification of cDCs in the spleens of WT mice maintained in either SPF or germ-free conditions and in SPF mice treated with broad-spectrum antibiotics. n = 13 SPF mice, 8 germ-free mice, and 10 antibiotic-treated mice; at least two experiments per condition; two-way ANOVA with Sidak’s multiple comparisons post-test. (D) Representative strategy for the identification of subset-committed pre-cDCs (a bone marrow sample is shown) by flow cytometry and quantification of pre-cDC1s and pre-cDC2s in the bone marrow and spleen of WT mice at different ages of post-natal development. Pre-cDCs are defined as Lineage(CD3e/CD19/NK1.1/Ter119)⁻B220⁻Ly6G⁻CD11c⁺Flt3⁺Sirpα^low cells; committed pre-cDC1s are SiglecH⁻Ly6C⁻, whereas committed are pre-cDC2 SiglecH⁻Ly6C⁺. n = 5, day 2 samples; 7, day 7 samples; 11, day 14 samples; 13, day 21 samples; 6, day 28 samples; and 4 adult samples; data verified in at least two different experiments per time point; two-way ANOVA with Sidak’s multiple comparisons post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.