Livecyte kinetic cytometer combined with RpHluorin2 reporter system for real-time single-cell resolution monitoring of JTC-801-induced alkaliptosis.

Intracellular alkalization-driven alkaliptosis has emerged as a promising therapeutic target for cancer intervention, yet static endpoint detection limits insights into its spatiotemporal dynamics. Here, we integrated the Livecyte Kinetic Cytometer (ptychographic quantitative phase imaging, QPI) with the RpHluorin2 pH-sensitive fluorescent reporter system to establish a real-time, single-cell resolution platform for monitoring JTC-801-induced alkaliptosis in human bladder cancer 5637 cells. The Livecyte system enabled continuous 6-hour tracking of RpHluorin2 fluorescence dynamics and cellular biophysical parameters at a 5-minute temporal resolution. JTC-801 induced a dose- and time-dependent reduction in RpHluorin2 fluorescence (a signal consistent with intracellular alkalization) and a concomitant impairment of key cellular motility parameters, including displacement, migration speed, and movement directionality. This integrated platform overcomes the limitations of traditional endpoint assays, enabling holistic characterization of alkaliptosis dynamics at the single-cell level, and provides a useful technical tool for the discovery and development of pH-targeted anticancer therapeutics. • A real-time, single-cell platform that couples label-free quantitative phase imaging (Livecyte) with the RpHluorin2 pH reporter to monitor alkaliptosis. • Continuous, correlated readout of intracellular pH (via fluorescence) together with cellular biophysical and motility parameters during JTC-801 treatment. • A reproducible tool for dissecting pH-dependent cell death dynamics and for screening pH-targeted anticancer agents.