Optimizing the Seahorse XF Mito Stress Test Workflow and Troubleshooting Notes: A Stepwise Protocol for HUVECs

This protocol details an optimized step-by-step procedure for performing the Seahorse XF Cell Mito Stress Test on human umbilical vein endothelial cells (HUVECs) using the Agilent Seahorse XF Pro Analyzer. Designed to address practical challenges often overlooked in standard manuals, the method preserves the native adherent state of HUVECs—a key in vitro model in vascular aging (VA) research—enabling real-time, label-free measurement of mitochondrial respiration and glycolytic function without cell detachment. The workflow is presented chronologically, covering instrument preparation, cell seeding, compound loading, assay execution, and post-assay normalization, with integrated notes and troubleshooting tips refined through hands-on experience based on the official manuals. This protocol aims to set up a detailed, rearranged standard workflow to improve experimental efficiency, reduce operator error, and support reproducible and well-organized metabolic profiling of HUVECs in aging and cardiovascular studies.