Cadosch Dieter, Meagher James, Gautschi Oliver P, Filgueira Luis
School of Anatomy and Human Biology, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
J Neurosci Methods. 2009 Mar 30;178(1):182-7. doi: 10.1016/j.jneumeth.2008.12.008. Epub 2008 Dec 14.
The attempt to visualise intracellular protein metal complexes has currently been difficult due to the unavailability of probes for such molecular structures. Newport Green DCF diacetate ester is a cell permeant acetate ester, which becomes fluorescent after hydrolysis. This molecule is initially uncharged, allowing it to pass through cell membranes. Once in the cell, it is hydrolysed and becomes charged, hindering its escape from the cell and allowing it to bind charged protein metal complexes, which then become fluorescent.
In this study, we exposed cultured human monocyte-derived dendritic cells (mDC) to a variety of metal ions with the aim of having the cells take up and process protein metal complexes. Newport Green DCF diacetate ester was used to fluorescently label intracellular protein metal complexes.
Flow cytometry analysis and confocal imaging showed specific staining for mDC exposed to aluminium, chromium, nickel, titanium and zirconium ions. The intensity of staining varied between ion types, whereby Ti(III) resulted in the brightest fluorescence signal. Aluminium, Cr(III), Ni, Ti(IV) and Zr(IV) were also clearly detectable.
For the first time, intracellular metal ion protein complexes undergoing cellular processing were successfully visualised in human mDC using flow cytometry and confocal microscopy.
由于缺乏针对此类分子结构的探针，目前可视化细胞内蛋白质金属复合物的尝试一直很困难。纽波特绿DCF二乙酸酯是一种细胞可渗透的乙酸酯，水解后会发出荧光。该分子最初不带电荷，使其能够穿过细胞膜。一旦进入细胞，它就会水解并带上电荷，阻碍其从细胞中逸出，并使其能够结合带电荷的蛋白质金属复合物，然后这些复合物会发出荧光。
在本研究中，我们将培养的人单核细胞衍生树突状细胞（mDC）暴露于多种金属离子，目的是让细胞摄取和处理蛋白质金属复合物。纽波特绿DCF二乙酸酯用于对细胞内蛋白质金属复合物进行荧光标记。
流式细胞术分析和共聚焦成像显示，暴露于铝、铬、镍、钛和锆离子的mDC有特异性染色。染色强度因离子类型而异，其中Ti(III)产生最亮的荧光信号。铝、Cr(III)、镍、Ti(IV)和Zr(IV)也清晰可检测到。
首次使用流式细胞术和共聚焦显微镜在人mDC中成功可视化了正在进行细胞处理的细胞内金属离子蛋白质复合物。
