Coleman Jack, Xiang Yuejun, Pande Praveen, Shen Dee, Gatica Divina, Patton Wayne F
R&D, Enzo Life Sciences, Farmingdale, New York 11735, USA.
J Biomol Screen. 2010 Apr;15(4):398-405. doi: 10.1177/1087057110364242. Epub 2010 Mar 17.
Lysosomes are membrane-bound subcellular organelles involved in the degradation of macromolecules and pathogens in diverse processes, including endocytosis, phagocytosis, and autophagy. A red fluorescent probe was developed that is selectively sequestered in acidic organelles. U20S cells pretreated with 64 microM chloroquine for as little as 5 h show a dramatic increase in lysosome-like vesicle number and volume. The probe can be employed for highlighting lysosome-like organelles under conditions wherein cells produce vacuoles that contain most of the degradative enzymes of the lysosome but are not as acidic as the parent organelle. Using a conventional fluorescence microplate reader, the half-maximal effective concentration (EC(50)) of chloroquine was estimated. The high Z' score obtained using the assay demonstrated excellent signal-to-noise ratios. The fluorescence microplate assay was successfully employed to screen a small-molecule compound library for agents that increase lysosomal volume and number. One potential application of the new assay is in the toxicology portion of preclinical drug safety assessment (ADME-Tox) workflows, using in vitro cell culture models to aid in the drug development process.
溶酶体是一种膜结合的亚细胞细胞器，参与多种过程中大分子和病原体的降解，包括内吞作用、吞噬作用和自噬作用。开发了一种红色荧光探针，它能选择性地被隔离在酸性细胞器中。用64微摩尔氯喹预处理U20S细胞仅5小时，溶酶体样囊泡的数量和体积就会显著增加。该探针可用于在细胞产生含有溶酶体大部分降解酶但酸性不如母细胞器的液泡的条件下突出显示溶酶体样细胞器。使用传统的荧光酶标仪，估计了氯喹的半数有效浓度（EC50）。使用该测定法获得的高Z'分数表明具有出色的信噪比。荧光酶标法成功用于筛选小分子化合物库，以寻找增加溶酶体体积和数量的试剂。新测定法的一个潜在应用是在临床前药物安全性评估（ADME-Tox）工作流程的毒理学部分，使用体外细胞培养模型来辅助药物开发过程。
