Flow Cytometry Protocols

Cornelis J. H. Pronk, David Bryder

Myeloerythroid-restricted precursor cells, derived from multipotent hematopoietic stem cells, 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 be able to study and understand the underlying mechanisms that guide various cell fate decisions. Also, this approach opens for 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. A wealth of literature has demonstrated the feasibility of similar approaches also for the human system. However, in this chapter, we focus on the identification of bone marrow cells derived from C57BL/6 mice, in which flow cytometry-based immunophenotypic applications have been most widely developed. This should allow also 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 flow cytometer with factory standard configuration.