Protocol for real-time measurement of mitochondrial bioenergetics in 3D-cultured brain tumor stem cells using the Resipher system.

Aberrant mitochondrial function can lead to severe human diseases, including neurodegenerative diseases and cancer. Here, we describe a cell-based protocol for measuring different mitochondrial respiratory parameters using the high-resolution real-time Resipher system. We optimized this protocol on brain tumor stem cells cultured in three-dimensional spheroids. We provide essential optimization steps for cell seeding density, mitochondrial respiration modulator concentrations, running the assay, and data analysis. For complete details on the use and execution of this protocol, please refer to Burban et al.¹.