EBIs and EBI macrophages are increased following administration of Epo, permitting characterization of EBI macrophages by CITE-seq. IFC analysis of EBIs from saline solution–treated (A) vs Epo-treated (B) mouse BM demonstrates an increase not only in the size of EBIs (increased CD71⁺ area) but also in the number of EBIs after Epo stimulation, indicating a parallel increase in the number of EBI macrophages. Each point represents an EBI observed by IFC; all EBIs observed in one biological replicate are shown. Spearman correlation coefficient r values are shown on the graphs (P < .0001). (C) The number of EBIs after Epo stimulation increased approximately 4-fold by IFC evaluation (n = 4 biologic repeats, mean ± SD shown in the bar graph; ∗∗P = .002 comparing raw values with unpaired Student t test). (D) Ratio of CD11b⁺:CD71⁺ area within each EBI (median shown in graph; ∗∗∗∗P < .0001 by Mann-Whitney test). (E,F) ICGS2 (Iterative Clustering and Guide-gene selection version 2) analysis and CellHarmony of single-cell CITE-seq data collected from the BM clusters enriched in EBIs of saline solution–treated (E) and Epo-treated (F) BM reveals 28 distinct clusters. Clusters 4 and 20 are composed of early erythroblasts remaining despite Ter119⁺ depletion; clusters 18, 22, 33, 39, and 12 are granulocyte precursors that were also not completely removed despite depletion for Ly6G; and clusters 9, 13, 15, 23, and 19 have a transcriptome compatible with macrophage/monocyte lineage. Cluster 7 demonstrates transcriptomic characteristics of plasmacytoid dendritic cells, such as Siglech, Bst2, and Ly6d, and therefore is not considered a macrophage subset. (G) Fold change in relative frequencies of the cells in the macrophage/monocyte, erythroid, and granulocytic clusters at baseline vs with Epo treatment as indicated by percentage of captured cells in each sample.