Erythroid maturation has been classically defined based on the remarkable changes visualized through microscopy. These involve the decrease in cell size, nuclear condensation and organelle loss, and include the final unique asymmetric division creating the short-lived nucleated pyrenocyte and the enucleate reticulocyte that matures into the red blood cell. Understanding the regulation of these processes has been challenging due to the difficulty in obtaining sufficient numbers of cells, particularly of rare intermediates, to study by microscopy. While flow cytometry can provide quantitative analysis of high cell numbers as well as critical tools for assaying processes like cell cycle, apoptosis and cell signaling, it cannot analyze or categorize cells based on morphology. Imaging flow cytometry (IFC) combines microscopy and flow cytometry by capturing brightfield and fluorescent images of large numbers of cells, which can be quantitated for both morphometric and fluorescent characteristics. Over the past 10 years, this approach has been increasingly used to study aspects of erythropoiesis. This chapter describes how to utilize IFC to enumerate multiple specific stages of erythropoiesis from primary tissue, as well as how to culture primary progenitors to enrich for the rare late stage enucleating cells in order to examine intracellular proteins involved in enucleation. These methods demonstrate the approaches and strength of IFC as a tool to bridge the power of microscopy and flow cytometry to more fully interrogate erythropoiesis.
Keywords: Bone marrow; Enucleation; Erythroblast; Erythropoiesis; ImageStream; Imaging flow cytometry.