Changing the balance of granulocyte and erythrocyte production within EBIs. Each point represents an EBI observed by IFC; all EBIs observed in one biological replicate are shown. (A) Administration of GCSF leads to an increase in CD11b⁺ cells within the EBIs along with a decrease in the number of BM EBIs that contain 2 or more erythroblasts per cluster. Plots of CD11b⁺ area vs CD71⁺ area measured by IFC for all EBIs in a representative experiment for each condition are shown. In the case of 250 μg/kg GCSF treatment which dramatically suppressed medullary erythropoiesis, clusters with 3 CD71⁺ cells were rare because of the overall paucity of erythroblasts in the BM, so clusters with just 2 CD71⁺ cells were considered as EBIs in this analysis. Spearman correlation coefficient r values are shown on the graphs (P < .0001). (B) Quantification of the slope of CD11b⁺ vs CD71⁺ area in control and GCSF-injected (n = 3, mean ± SD is shown in the bar graphs; ∗∗P < .01 based on unpaired Student t test). (C) Ratio of CD11b:CD71 area within each EBI, with the line representing the median CD11b⁺:CD71⁺ area ratio (∗∗∗∗P < .0001 by Mann-Whitney test). (D) EBIs from Gfi1^−/− mice, which have an arrest in early granulopoiesis, show the reverse trend as imaged by IFC, with fewer CD11b⁺ cells and a predominance of CD71⁺ cells within the EMBIs. Of note, these mice were analyzed at 8 to 9 weeks of age because of the early mortality associated with complete deficiency of Gfi1. (E) Quantification of the slope of CD11b⁺ vs CD71⁺ area in control and Gfi1^−/− mice (n = 3-4 biologic repeats per condition as shown, mean ± SD is shown in the bar graphs; ∗P = .0306 based on unpaired Student t test). (F) Ratio of CD11b⁺:CD71⁺ area within each EBI, with the line representing the median CD11b:CD71 area ratio (∗∗∗∗P < .0001 by Mann-Whitney test).