Flow Cytometry-Based Identification of Immature Myeloerythroid Development

Precursor cells of the myeloerythroid cell lineages give rise to mature cells of the granulocyte, monocyte, erythroid, and/or thrombocytic lineages. High-resolution profiling of the developmental stages, from hematopoietic stem cells to mature progeny, is important to study and understand the underlying mechanisms that guide various cell fate decisions. In addition, this approach provides greater insights into pathogenic events such as leukemia, diseases that are most often characterized by halted differentiation at defined immature precursor levels. In this chapter, we provide protocols and discuss approaches concerning the analysis and purification of immature myeloerythroid lineages by multiparameter flow cytometry. Although recent data have demonstrated the feasibility of similar approaches also for the human system, we will focus our chapter on C57BL/6 mice, in which immunophenotypic applications have been most widely developed. This should also allow for its application in genetically modified models on this background. For maximal reproducibility, all protocols described have been established using reagents from commercial vendors to be analyzed on a three-laser flow cytometer with factory standard configuration.