Duszyc Kinga, Noordstra Ivar, Yap Alpha S, Gomez Guillermo A
Divisions of Cell and Developmental Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, 4072, Australia.
Bio Protoc. 2021 Nov 20;11(22):e4232. doi: 10.21769/BioProtoc.4232.
Apoptotic cell death eliminates unhealthy cells and maintains homeostatic cell numbers within tissues. Epithelia, which serve as fundamental tissue barriers for the body, depend on a physical expulsion of dying cells (apoptotic cell extrusion) to remain sealed and intact. Apoptotic cell extrusion has been widely studied over recent years, with researchers using various approaches to induce apoptotic cell death. Unfortunately, the majority of chemical-based approaches for cell death induction rely on sporadically occurring apoptosis and extrusion, making imagining lengthy, often unsuccessful, and difficult to capture in high-quality images because of the frequent frame sampling needed to visualise the key molecular processes that drive extrusion. Here, we present a protocol that describes steps needed for laser-mediated induction of apoptosis in a cell of choice, followed by imaging of apoptotic extrusion in confluent monolayers of epithelial cells. Moreover, we provide the description of a new approach involving the mixing of labelled and unlabelled cells. In particular, this protocol characterises how cells surrounding apoptotic cells behave, with high spatial and temporal resolution. This can be achieved without the optical interference that apoptotic cells cause as they are physically expelled from the monolayer and move out of focus for imaging. Finally, the protocol is accompanied by detailed procedures describing cell preparation for apoptotic extrusion experiments, as well as post-acquisition analysis required to evaluate rates of successful extrusion.
凋亡性细胞死亡可清除不健康细胞，并维持组织内细胞数量的稳态。上皮组织作为身体的基本组织屏障，依赖于对垂死细胞的物理排出（凋亡性细胞挤出）来保持密封和完整。近年来，凋亡性细胞挤出受到了广泛研究，研究人员使用各种方法诱导凋亡性细胞死亡。不幸的是，大多数基于化学的细胞死亡诱导方法依赖于偶尔发生的凋亡和挤出，使得成像过程漫长，往往不成功，并且由于可视化驱动挤出的关键分子过程所需的频繁帧采样，难以获得高质量图像。在这里，我们提出了一种方案，该方案描述了在选定细胞中激光介导诱导凋亡所需的步骤，随后对汇合单层上皮细胞中的凋亡性挤出进行成像。此外，我们还介绍了一种涉及标记细胞和未标记细胞混合的新方法。特别是，该方案以高空间和时间分辨率表征了凋亡细胞周围的细胞行为。这可以在凋亡细胞从单层中被物理排出并移出成像焦点时不会产生光学干扰的情况下实现。最后，该方案还附带了详细的程序，描述了凋亡性挤出实验的细胞制备以及评估成功挤出率所需的采集后分析。
