An Organoid-Based Assay for Evaluating Oxidative Stress-Reactive Fluorescent Probes Using the CERO 3D Bioreactor in a...

Oxidative stress is a central driver of acute liver injury, and protein carbonylation and lipid aldehyde formation are among its most direct chemical signatures. Most carbonyl-reactive fluorescent probes have been characterized only in two-dimensional culture, and their behavior in three-dimensional tissue is often unclear. Here we describe a protocol for evaluating carbonyl-reactive fluorescent probes in mouse hepatic organoids grown in suspension in the CERO 3D bioreactor, using acetaminophen (APAP) overdose as a well-established model of acute oxidative liver injury. We demonstrate the assay with P7-Hz, a hydrazine-based probe that reacts with carbonyl groups on oxidatively damaged proteins and lipids to form the fluorescent hydrazone product P7-Hz-Ali. The protocol covers organoid culture and maintenance, APAP dosing to induce injury, probe incubation (labeling), and imaging-based quantification of the fluorescence signal as a readout of carbonylation, together with co-staining viability to distinguish probe-specific signal from cytotoxicity. Although P7-Hz is used as the demonstration probe, the same workflow accommodates other carbonyl-reactive probes whose hydrazone (or analogous) product is spectroscopically distinguishable from the unreacted probe, providing a reproducible three-dimensional platform for probe validation.