Buschhaus Johanna M, Gibbons Anne E, Luker Kathryn E, Luker Gary D
Center for Molecular Imaging, Department of Radiology, University of Michigan, Ann Arbor, Michigan.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan.
Curr Protoc Cell Biol. 2017 Dec 11;77:21.12.1-21.12.12. doi: 10.1002/cpcb.36.
Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments. Apoptosis of breast cancer cells in 2D culture, spheroids, and in vivo murine breast tumor xenografts in response to a variety of genetic and pharmacologic methods implicated in apoptosis of cancer cells was studied. This approach for quantifying apoptosis of cancer cells is based on caspase-3 activity at single-cell resolution using FLIM. © 2017 by John Wiley & Sons, Inc.
半胱天冬酶-3是一种蛋白水解酶，在细胞凋亡性死亡中起关键效应作用。测定半胱天冬酶-3的活性可提供有关癌细胞活力及对治疗反应的关键信息。为了在完整细胞和活体小鼠中检测细胞凋亡，使用了一种通过Förster共振能量转移（FRET）检测半胱天冬酶-3活性的荧光成像报告基因。通过荧光寿命成像显微镜（FLIM）测量FRET的变化。与基于荧光强度的FRET测量不同，寿命测量与报告基因浓度及组织中的光散射无关，这使得FLIM成为在三维环境中成像的可靠方法。研究了二维培养的乳腺癌细胞、球体以及体内小鼠乳腺肿瘤异种移植瘤在多种与癌细胞凋亡相关的基因和药理学方法作用下的凋亡情况。这种定量癌细胞凋亡的方法基于使用FLIM在单细胞分辨率下的半胱天冬酶-3活性。© 2017约翰威立国际出版公司。
