热原性死亡（pyroptosis）已被确认为一种炎症性的程序化细胞死亡形式。它可以通过不同的刺激因素诱发，包括病原体侵入或细胞压力/危险信号的释放，在溶解过程中释放数百种蛋白质，并引起邻近细胞的复杂反应。热原性死亡是由气细胞膜（GSDM）蛋白家族执行的，它们在被半胱氨酸蛋白酶剪切后形成跨膜孔，释放细胞因子诱导炎症。然而，尽管GSDM蛋白在炎症性疾病和癌症的发展中非常重要，但该细胞死亡途径的下游后果仍有很多待解。目前，传统方法，如药物治疗或化学强制聚集，受限于对细胞人群热原性死亡信号进行时空分析，因为所有细胞都被激发为发生热原性死亡。在本文中，我们提供了一种新型光遗传学工具NLS_PhoCl_N-GSDMD_mCherry的应用方案，该方案在共聚焦显微镜设置中实现了精确的时空热原性死亡诱导，随后对细胞死亡过程进行成像并对实验进行定量分析。该工具为研究热原性死亡的激活及其对旁观细胞反应的影响提供了新的机会。© 2023. 作者等专属授权给Springer Science+Business Media, LLC，Springer Nature的一部分。

Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.