Journal article

Authors list: Ross, R; Grimmel, J; Goedicke, S; Möbus, AM; Bulau, AM; Bufler, P; Ali, S; Martin, MU

Publication year: 2013

Pages: 219-227

Journal: Journal of Immunological Methods

Volume number: 387

Issue number: 1-2

ISSN: 0022-1759

DOI Link: https://doi.org/10.1016/j.jim.2012.10.017

Publisher: Elsevier

Abstract: 
The dual function cytokines IL-1 alpha, IL-33 and IL-37 are members of the IL-1 cytokine family. Besides of being able to bind to their cognate receptors on target cells, they can act intracellularly in the producing cell. All three are able to translocate to the nucleus and have been discussed to affect gene expression. In order to compare and quantitate nuclear translocation of these IL-1 family members we established a robust technique which enables to measure nuclear localization on a single cell level by flow cytometry. Vectors encoding fusion proteins of different IL-1 family members with enhanced green fluorescent protein were cloned and cell lines transiently transfected with these. Fluorescent fusion proteins in intact cells or in isolated nuclei were detected subsequently by fluorescence microscopy and flow cytometry, respectively. Depending on the cellular system, cells and nuclei were distinguishable by flow cytometry in forward scatter/sideward scatter. Fluorescent fusion proteins were detectable in isolated nuclei up to three days following preparation. Signal intensity of fusion proteins of IL-33 and IL-37 in isolated nuclei but not of IL-1a, was markedly increased by fixation with paraformaldehyde, directly following cell lysis, indicating that IL-1 alpha binds stronger to nuclear structures than IL-33 and IL-37. Nuclear translocation of fluorescent IL-37 fusion proteins in a stably transfected RAW264.7 mouse macrophage cell line required stimulation with lipopolysaccharide. Applying this method we demonstrated that a prolonged lag phase of more than 15 h before LPS-stimulated nuclear translocation was detected. In summary, we present a robust method to analyze and quantitate nuclear localization of IL-1 cytokine family members. (C) 2012 Elsevier B.V. All rights reserved.

Harvard Citation style: Ross, R., Grimmel, J., Goedicke, S., Möbus, A., Bulau, A., Bufler, P., et al. (2013) Analysis of nuclear localization of interleukin-1 family cytokines by flow cytometry, Journal of Immunological Methods, 387(1-2), pp. 219-227. https://doi.org/10.1016/j.jim.2012.10.017

APA Citation style: Ross, R., Grimmel, J., Goedicke, S., Möbus, A., Bulau, A., Bufler, P., Ali, S., & Martin, M. (2013). Analysis of nuclear localization of interleukin-1 family cytokines by flow cytometry. Journal of Immunological Methods. 387(1-2), 219-227. https://doi.org/10.1016/j.jim.2012.10.017