Herrera Sara Abad, Grifell-Junyent Marta, Pomorski Thomas Günther
Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.
Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark.
Bio Protoc. 2022 Feb 20;12(4):e4330. doi: 10.21769/BioProtoc.4330.
All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their substrate specificity, and their regulation remain to be elucidated. Here, we describe an optimized quantitative flow cytometry-based lipid uptake assay utilizing nitrobenzoxadiazolyl (NBD) fluorescent lipids to study lipid internalization in mammalian cell lines, which allows characterizing lipid transporter activities at the plasma membrane. This approach allows for a rapid analysis of large cell populations, thereby greatly reducing sampling variability. The protocol can be applied to study a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane, and to uncover the dynamics of lipid transport at the plasma membrane. Graphic abstract: Internalization of NBD-labeled lipids from the plasma membrane of CHO-K1 cells.
所有真核细胞都配备有跨膜脂质转运蛋白，它们是膜脂质不对称、囊泡运输和膜融合的关键参与者。这些转运蛋白的突变与人类疾病之间的联系凸显了它们在细胞稳态中的重要作用。然而，它们活动的许多关键特征、底物特异性及其调节仍有待阐明。在这里，我们描述了一种基于优化的定量流式细胞术的脂质摄取测定法，该方法利用硝基苯并恶二唑基（NBD）荧光脂质来研究哺乳动物细胞系中的脂质内化，从而能够表征质膜上脂质转运蛋白的活性。这种方法允许对大量细胞群体进行快速分析，从而大大降低采样变异性。该方案可用于研究多种哺乳动物细胞系，测试基因敲除对质膜脂质内化的影响，并揭示质膜脂质运输的动态过程。图形摘要：CHO-K1细胞质膜上NBD标记脂质的内化。
