Figure 2.

Gating strategy of erythroblastic islands (EBIs) for analysis by imaging flow cytometry (IFC). (A) Left panel: multiplets (cell clusters) characterized by their large area in brightfield (BF) are gated. Right panel: the cell clusters are then plotted as per intensity of F4/80 and of CD71 stain. The gate containing clusters high in fluorescence intensity for both F4/80 and CD71 is enriched in EBIs (gate). Manual inspection of the cell clusters in the EBI gate allows confirmation of the events (tagged yellow) that clearly have the structure of an EBI. Some EBIs are located outside of the EBI gate but with decreased frequency. (B) Examples of EBIs (representative of the yellow-marked events within the EBI gate) characterized by a central F4/80⁺ macrophage surrounded by CD71⁺ and/or Ter119⁺ erythroblasts. An anucleate reticulocyte (arrow) is occasionally seen, still in contact with the macrophage. (C) Examples of cell clusters, which although within the EBI gate (double positive for F4/80 and CD71), do not have the classic appearance of an intact EBI. These events would have been included in the analysis by “blind” flow cytometry, potentially leading to misguided conclusions about EBI macrophage markers. (D) Representative examples of EBIs isolated from mouse spleen after development of stress erythropoiesis (4 days post-induction of anemia with phlebotomy of 500 µL of blood). (E) Representative examples of EBIs isolated from mouse fetal livers (E13.5).