Ritter Portia, Yousefi Keyvan, Ramirez Juliana, Dykxhoorn Derek M, Mendez Armando J, Shehadeh Lina A
Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine; Department of Medicine, Division of Cardiology, University of Miami Leonard M. Miller School of Medicine.
Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine; Department of Molecular and Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine.
J Vis Exp. 2018 Nov 17(141). doi: 10.3791/58564.
The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.
通过低密度脂蛋白受体（LDLR）介导的内吞作用对低密度脂蛋白胆固醇摄取的调节是包括代谢紊乱、心血管疾病和肾脏疾病在内的各种主要病理学中一个重要的研究领域。目前，尚无可用方法在监测细胞健康状况的同时评估低密度脂蛋白摄取。本研究提出了一种方案，使用活细胞成像分析系统，在监测细胞健康状况的同时获取低密度脂蛋白流入的系列测量值。这种新技术在三种人类细胞系（肝细胞、肾小管上皮细胞和冠状动脉内皮细胞）中进行了长达四小时的时间进程测试。此外，该技术的灵敏度通过众所周知的低密度脂蛋白摄取抑制剂Dynasore和重组PCSK9蛋白以及低密度脂蛋白摄取促进剂辛伐他汀进行了验证。综上所述，该方法提供了一个中到高通量的平台，用于同时筛选药理活性以及监测细胞形态，从而监测调节低密度脂蛋白流入的化合物的细胞毒性。该分析可与不同的成像系统和分析软件一起使用。
