Gene expression profiling of newly generated and mature splenic EYFP⁺PCs from mice. Splenic newly generated EYFP⁺B220⁻ PCs were sorted 6 days after a third immunization with SRBCs (day 6 [D6]/boost), and splenic mature EYFP⁺B220⁻ PCs 3 months after the second immunization (M3/controls) (see protocol in supplemental Figure 2A). About 10 000 EYFP⁺ PCs per mouse were analyzed (D6, n = 3; M3, n = 4). (A) Sorting strategy of mature splenic EYFP⁺ B220⁻ PCs by use of a 2-step procedure. (B) Total number of EYFP⁺B220⁻ PCs in controls (M3) or boosted (D6) mice. (C) Heatmap of genes selected from the supervised microarray comparison of newly generated (D6) vs mature PCs (M3) in the mouse spleen (223 genes with fold change in expression >4 or <0.25, P < .05). The heatmap is based on normalized log₂ intensities that were probe-wise mean-centered across the 2 compared conditions. Columns represent individual samples, and rows represent specific gene probes, with upregulated genes in red and downregulated genes in blue. Genes selected for the single-cell analysis are highlighted. (D) Functional analysis of the 174 upregulated genes in mature vs newly generated PCs (more than fourfold upregulated; P < .05). The number of genes in each functional category is shown.