Real-Time Quantification of Cell Internalization Kinetics by Bioluminescent Probes.
Real-Time Quantification of Cell Internalization Kinetics by Bioluminescent Probes.
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通过生物发光探针实时定量细胞内化动力学。
DOI：
10.1007/978-1-0716-2473-9_7
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Wen,Jing
中科院分区：
文献类型：
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作者：
Castillo,Roxanne;Wu,Di;Cao,Zheng;Yan,Ran;Fajardo,Kalea;Ren,Jie;Lu,Yunfeng;Wen,Jing
除了细胞内稳态所需的营养物质的细胞内运输外，还在努力将蛋白质和基因等物质有效地输送到细胞中，用于治疗，基因编辑，疾病诊断等。为了评估这些物质的细胞内递送，常规方法对细胞内化的动态过程进行半定量和离散测量。在这里，我们详细的方法来量化细胞内化动力学的实时使用单独的纳米封装的生物发光萤火虫荧光素酶（FLuc）作为探针。我们包括一个全面的协议来合成和表征封装的FLuc，测定细胞中的实时生物发光（BL），并分析实时BL曲线以提取细胞内化动力学的关键参数。定量细胞内递送的动力学提供了解决控制膜易位的潜在机制的机会，并提供反映细胞状态和代谢的措施，同时在有效载体的临床开发中发挥关键作用。
Alongside the intracellular transport of nutrients needed for cellular homeostasis, great efforts exist to effectively deliver substances such as proteins and genes into the cell for therapy, gene editing, disease diagnosis, and more. To evaluate the intracellular delivery of such substances, conventional methods impose semi-quantifications and discrete measures of the dynamic process of cellular internalization. Herein, we detail the methods to quantify cell internalization kinetics in real-time using individually nano-encapsulated bioluminescent Firefly Luciferase (FLuc) enzymes as probes. We include a comprehensive protocol to synthesize and characterize the encapsulated FLuc, assay the real-time bioluminescence (BL) in cells, and analyze the real-time BL profile to extract key parameters of cell internalization kinetics. Quantifying the kinetics of intracellular delivery offers the opportunity to resolve the underlying mechanisms governing membrane translocation and provide measures reflecting cellular state and metabolism while playing a critical role in the clinical development of effective vectors.