Isolation of neutrophil precursors from bone marrow for biochemical and transcriptional analysis

Abstract

The neutrophilic granulocyte is the most numerous leukocyte in peripheral blood. The development from a multipotent progenitor cell to a mature neutrophil takes place in the bone marrow over a period of 10–14 days. In order to understand the cellular mechanisms behind this process, it is necessary to investigate cells from different stages of neutrophil differentiation. As no human cell line has the ability to faithfully reproduce the entire differentiation process from promyelocyte to segmented neutrophil the analysis of many maturation-dependent processes has to be done on neutrophil precursors from human bone marrow. For this purpose, a technique whereby neutrophil precursors can be isolated from the bone marrow and separated according to their maturity is required. Two different methods have been shown to be useful for isolation of immature neutrophils: density centrifugation on a Percoll gradient, where the increasing density of the cells with maturity forms the basis of the separation, and multidimensional flow cytometry, where a combination of size, granulation, and surface markers are used for the discrimination of different neutrophil precursors. This paper will review these two methods for separation of neutrophil precursors with special emphasis on Percoll density centrifugation and the use of cells isolated by this technique for the analysis of neutrophil-specific mRNAs and the biosynthesis of neutrophil granule proteins.