Selcen Selen, Wieland Lena, De Oliveira Tiago, Ghadimi Michael, Conradi Lena-Christin, Schneider Günter, Witte Leonie, Wirth Matthias
Department of General, Visceral and Pediatric Surgery, University Medical Center Göttingen, 37075, Göttingen, Germany.
Clinical Research Unit 5002, KFO5002, University Medical Center Göttingen, 37075, Göttingen, Germany.
Cell Death Discov. 2025 Aug 6;11(1):368. doi: 10.1038/s41420-025-02662-y.
Regulated cell death plays a central role in tissue homeostasis, disease progression, and therapeutic responses. However, tools to study these processes with high spatiotemporal resolution in physiologically relevant systems remain limited. Here, we present a fluorescent reporter cell system that enables real-time visualization of caspase-3/-7 activity via a DEVD-based biosensor, alongside a constitutive fluorescent marker for assessing successful transduction and cell presence. We generated stable cell lines expressing this reporter and adapted them to both 2D and 3D culture systems, including organoids. This platform allowed dynamic tracking of apoptotic events and viability loss at single-cell resolution. Using a proliferation dye, we also detected apoptosis-induced proliferation in neighboring cells. Furthermore, the system enabled simultaneous detection of immunogenic cell death via an endpoint measurement of surface calreticulin exposure by flow cytometry, supporting its application in studying immunogenic signaling. By measuring and integrating multiple cell death readouts by live-cell imaging, our system is well-suited for high-content screening and mechanistic dissection of different modes of cell death. When combined with complementary markers of pyroptosis and necroptosis, this platform may also be extended to investigate more complex, integrated forms of cell death.
程序性细胞死亡在组织稳态、疾病进展和治疗反应中起着核心作用。然而,在生理相关系统中以高时空分辨率研究这些过程的工具仍然有限。在此,我们展示了一种荧光报告细胞系统,该系统能够通过基于DEVD的生物传感器实时可视化半胱天冬酶-3/-7的活性,同时还有一个组成型荧光标记物用于评估成功转导和细胞存在情况。我们生成了表达该报告基因的稳定细胞系,并使其适用于二维和三维培养系统,包括类器官。这个平台允许在单细胞分辨率下动态跟踪凋亡事件和活力丧失。使用增殖染料,我们还检测到邻近细胞中凋亡诱导的增殖。此外,该系统能够通过流式细胞术对表面钙网蛋白暴露进行终点测量,同时检测免疫原性细胞死亡,支持其在研究免疫原性信号传导中的应用。通过活细胞成像测量和整合多种细胞死亡读数,我们的系统非常适合对不同细胞死亡模式进行高内涵筛选和机制剖析。当与细胞焦亡和坏死性凋亡的互补标记物结合时,这个平台也可以扩展到研究更复杂、整合形式的细胞死亡。
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