Imaging Flow Cytometric Analysis of Primary Bone Marrow Erythroblastic Islands

The erythroblastic island (EBI) is a multicellular functional erythropoietic unit comprising a central macrophage nurturing a rosette of maturing erythroblasts. Since the discovery of EBIs more than half a century ago, EBIs are still studied by traditional microscopy methods after enrichment by sedimentation. These isolation methods are not quantitative and do not enable precise quantification of EBI numbers or frequency in the bone marrow or spleen tissues. Conventional flow cytometric methods have enabled quantification of cell aggregates co-expressing macrophage and erythroblast markers; however, it is unknown whether these aggregates contain EBIs as these aggregates cannot be visually assessed for EBI content. Combining the strengths of both microscopy and flow cytometry methods, in this chapter we describe an imaging flow cytometry method to analyze and quantitatively measure EBIs from the mouse bone marrow. This method is adaptable to other tissues such as the spleen or to other species provided that fluorescent antibodies specific to macrophages and erythroblasts are available.