Fluorescent High-Content Imaging Allows the Discrimination and Quantitation of E-LDL-Induced Lipid Droplets and Ox-LDL-Generated Phospholipidosis in Human Macrophages
Fluorescent High-Content Imaging Allows the Discrimination and Quantitation of E-LDL-Induced Lipid Droplets and Ox-LDL-Generated Phospholipidosis in Human Macrophages
复制标题
DOI：
10.1002/cyto.a.20828
复制
发表时间：
2010-03-01
期刊：
影响因子：
3.7
通讯作者：
Schmitz, Gerd
中科院分区：
文献类型：
--
作者：
Grandl, Margot;Schmitz, Gerd
在摄取致动脉粥样硬化脂蛋白过程中形成的巨噬细胞泡沫细胞是动脉粥样硬化病变发展的标志。在这项研究中，人巨噬细胞与两种原型致动脉粥样硬化的LDL修饰酶降解LDL（E-LDL）和氧化LDL（Ox-LDL）从同一供体LDL制备。为了检测巨噬细胞脂质储存的差异，使用荧光高含量成像。使用Bodipy 493/503对脂滴进行染色，并使用荧光磷脂探针NBD-PE在高含量成像测定中检测内溶酶体磷脂病。使用磷脂质沉积诱导阳离子两亲性药物验证磷脂质沉积测定。此外，使用LipidTOX测定中性脂质和磷脂质病。用基于多通道激光的高容量共聚焦显微镜捕获96孔细胞培养微量滴定板的图像，随后分析基于细胞和孔的数据。加载E-LDL的巨噬细胞显示Bodipy 493/503和LipidTOX（TM）-绿色中性脂滴染色强度增加，与加载Ox-LDL和M-CSF分化的对照巨噬细胞相比，每个细胞的脂滴平均面积和数量更大。相反，与E-LDL-负载和M-CSF-分化的巨噬细胞相比，Ox-LDL-负载的巨噬细胞在内溶酶体区室中显示出NBD-PE和LipidTOX（TM）-Red可检测的磷脂质沉积的强度增加。过氧化物酶体增殖物激活受体-γ激动剂吡格列酮治疗导致脂滴诱导，这取决于巨噬细胞的脂质负荷状态。这些结果表明，E-LDL优先诱导脂滴，而Ox-LDL引起的人巨噬细胞内溶酶体磷脂质沉积，代表两种不同的脂质储存原则。因此，荧光高含量成像是一个有用的工具，以区分和量化的巨噬细胞中的脂质储存室也响应于影响细胞脂质代谢的药物。(C)2009年国际细胞计数促进学会
Macrophage foam cells formed during uptake of atherogenic lipoproteins are a hallmark of atherosclerotic lesion development. In this study, human macrophages were incubated with two prototypic atherogenic LDL modifications enzymatically degraded LDL (E-LDL) and oxidized LDL (Ox-LDL) prepared from the same donor LDL. To detect differences in macrophage lipid storage, fluorescent high-content imaging was used. Lipid droplets were stained using Bodipy 493/503, and the fluorescent phospholipid probe NBD-PE was used to detect endolysosomal phospholipidosis in high-content imaging assays. The phospholipidosis assay was validated using phospholipidosis-inducing cationic amphiphilic drugs. In addition, neutral lipids and phospholipidosis were determined using LipidTOX. Images of 96-well cell culture microtiter plates were captured with multichannel laser-based high-content confocal microscopy, and subsequently cell- and well-based data were analyzed. E-LDL-loaded macrophages show increased intensity of Bodipy 493/503 and LipidTOX(TM)-Green neutral lipid droplet staining and a greater mean area and number of lipid droplets per cell compared to Ox-LDL-loaded and M-CSF-differentiated control macrophages. In contrast, Ox-LDL-loaded macrophages show increased intensity of NBD-PE and LipidTOX(TM)-Red detectable phospholipidosis in the endolysosomal compartment compared to E-LDL-loaded and M-CSF-differentiated macrophages. Treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone leads to lipid droplet induction depending on the lipid loading state of the macrophages. These results indicate that E-LDL preferentially induces lipid droplets, while Ox-LDL provokes endolysosomal phospholipidosis in human macrophages representing two different lipid storage principles. Therefore, fluorescent high-content imaging is a useful tool to discriminate between and quantify lipid storage compartments in macrophages also in response to drugs affecting cellular lipid metabolism. (C) 2009 International Society for Advancement of Cytometry