Optimization and application of a flow cytometric PU.1 assay for murine immune cells

Journal of Immunological Methods
Greg NoelCora K Ogle

Abstract

PU.1 is a master transcription factor whose levels directly influence hematopoiesis, leukemia, susceptibility to sepsis, and macrophage function. Though measurement of PU.1 levels is important to health and disease, most studies have relied on PCR or western blots to measure the expression of this transcription factor. An accessible, validated assay that could measure PU.1 protein in subpopulations of cells is needed. In this work we present an optimized flow cytometric assay to detect PU.1 in subpopulations of immune cells. Murine myeloid cells were fixed in paraformaldehyde, permeabilized, and then stained with anti PU.1 in the presence and absence of a blocking peptide containing the binding site of the antibody. The bound anti PU.1 was then visualized with a labeled second antibody. Methanol and ethanol were tested for their relative ability to permeabilize cells and detect PU.1. The effect of the procedure upon the ability to detect cellular subpopulations was examined. Relative PU.1 1evels in normal T cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, and progenitors from the spleen and/or bone marrow were determined. Finally, PU.1 levels in proliferating myeloid cells from burn mice were determined. Th...Continue Reading

References

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Jan 20, 2005·The Journal of Experimental Medicine·Stephen L NuttLi Wu
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Citations

Dec 10, 2013·Experimental Hematology·Rachel ZiliottoRodney P DeKoter
Apr 17, 2020·FEBS Open Bio·Takuya YashiroChiharu Nishiyama

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