Dual-Anchoring Fluorescent Probes Enable Facile Recognition of Pyroptosis through Dynamic Tracking of Plasma Membrane

Pyroptosis is a pro-inflammatory form of programmed cell death mediated by the gasdermin protein family, often leading to potentially fatal systemic inflammation and associated with severe diseases like cancer. However, there remains a lack of simple yet effective tools for identifying and monitoring pyroptosis in real time. Herein, we report dual-anchoring fluorescent probes (SPD-R) designed for long-term tracking of the plasma membrane and demonstrate their utility in the identification and real-time monitoring of pyroptosis over hours. The identification of pyroptosis heavily relies on the dynamic changes of the plasma membrane during its distinct stages, making the key feature of these probes their ability to maintain long-term anchoring to the plasma membrane. In the designed SPD-R probes, three positive charges and a rigid steric hindrance unit were incorporated to enhance probe impermeability, while long alkyl chains at both ends were introduced to promote interactions with the lipid bilayer of the plasma membrane. These probes exhibit strong labeling capacity across various cell types and achieve up to several hours of stable anchoring on the plasma membrane, enabling real-time tracking of the entire pyroptosis process induced by LPS. The SPD-R probes successfully label and track significant increases in cell volume and notable bubbling during typical pyroptosis, providing a straightforward approach for pyroptosis recognition. Furthermore, this method was extended to the fast screening of Chinese medicines that induce cancer cell pyroptosis, with Hedyotis diffusa serving as an example.