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该文主要研究了如何定量检测完整细胞或细胞核中的IL-1家族（IL-1α、IL-33、IL-37），为核内细胞因子的检测提供了一个较好的参考方法。
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Analysis of nuclear localization of interleukin-1 family cytokines by flow cytometry
摘要：
The dual function cytokines IL-1α, 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 intracellularlyin 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 familymemberswe established a robust techniquewhich enables tomeasure 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 nucleiwere distinguishable by flowcytometry 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-1α, was markedly increased by fixation with paraformaldehyde, directly following cell lysis, indicating that IL-1α 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.
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发表于 2012-11-22 13:35:10
