Agilent Seahorse XF HS Mini‐Platform: A Comprehensive Guide for Real‐Time Analysis of Cellular Energy Metabolism

ABSTRACT The Agilent Seahorse XF HS Mini‐platform enables real‐time, label‐free measurement of cellular energy metabolism by simultaneously quantifying the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR). This guideline provides a detailed protocol for conducting mitochondrial stress tests, glycolytic rate assays, and real‐time ATP rate assay using the Seahorse XF HS Mini system, which has not been systematically demonstrated in previous literature. The methodology covers key steps including cell preparation, sensor cartridge hydration, assay medium formulation, compound loading, and data normalization. This guideline emphasizes experimental design, troubleshooting, and applications of the Seahorse XF Mini system in studying metabolic functions within the fields of food safety and health, metabolic syndrome disorders, and food science, as well as cancer, immunology, and neurodegenerative diseases. This guideline serves as an essential resource for researchers aiming to investigate cellular bioenergetics with high precision and reproducibility.