CD11b⁺ cells participate in BM EBIs in a constant ratio with erythroblasts. (A) BM clusters, enriched in EBIs, are collected by gravity sedimentation through a BSA gradient. (B) A population of large cell clusters is gated based on their large area in the bright field, which are then gated for double positivity for F4/80 and CD71; EBIs are then selected manually out of this gate after direct visualization.¹³ Upon visual inspection and identification of a cluster as an EBI (central F4/80⁺ macrophages surrounded with at least 3 CD71⁺ erythroblasts), the event is marked in yellow and designated to the manually tagged EBI population. (C) Representative images of EBIs from ImageStream analysis demonstrating varying ratios of CD11b to CD71. (D) Flow cytogram of CD11b⁺ area vs CD71⁺ area in the EBIs for one representative experiment. Each point represents an EBI observed by IFC. Upon visual inspection and identification of a cluster as an EBI, the event is marked in yellow and designated as a tagged EBI. All EBIs observed in one biological replicate are shown. Least-square linear regression was used to produce the dotted line, with the constraint to cross the point (X0, Y0), and therefore, because the intercept is 0, the slope represents the ratio of CD11b⁺ area to CD71⁺ area within EBIs. The average slope in 5 experiments was in a fairly narrow range of 0.690 ± 0.035, indicating a constant ratio of CD11b⁺ cells to erythroblasts in the EBIs and implying a physiologic significance. Spearman correlation coefficient r values for all replicates ranged from 0.4 to 0.6 (P < .0001). (E) Whole-mount immunofluorescence staining of intact BM shows cells stained for CD11b (blue) and CD71 (red) intimately associated with an F4/80⁺ macrophage (green) in situ. Image shown is a maximum-intensity projection of a confocal Z-stack. Scale bar represents 10 μm.