An optimized permeabilization step for flow cytometry analysis of nuclear proteins in myeloid differentiation of blood cells into neutrophils

Abstract

Polymorphonuclear leukocytes (PMNLs) or neutrophils play an important role in the innate immune response. Working with human neutrophils is challenging because these cells are sensitive to changes in the surrounding media and quickly become apoptotic. Meanwhile the experiments with mature neutrophils may be very important for studies of blood function. In this paper we propose an improved technique of flow cytometry nuclear protein analysis with double antibody labeling, which allows direct comparison of protein quantity (overlay histograms) in the primary cells (neutrophils) and progenitor cell lines (line HL-60), to study differentiation process and for other research purposes. We suggest improved technique to analyze and compare nuclear proteins levels in the myeloid differentiation model system (HL-60 cell line) and / or primary human neutrophils. This method was justified with measurement of GFI1 protein expression level, as well-known transcription factor, typical and essential for mature neutrophils.The key protocol features are as follows: • Suggested protocol allows simply, direct and correct visual comparison of flow cytometry data in overlay diagrams for myeloid blood cells on various stages of differentiation. • 70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better peak resolution than MeOH and Saponin permeabilization. • Non-specific antibody binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat serum. Method name: Flow cytometry, Keywords: Polymorphonuclear leukocytes, Neutrophils, HL-60, Flow cytometry, Protein detection, Permeabilization