Protocol for Seahorse 3D Mito Stress assay in patient-derived pediatric brain tumor single neurospheres.
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UNLABELLED: Metabolism is essential for cellular functions and is often altered in cancer. While Seahorse assays are well established for measuring metabolic changes in 2D cell cultures, their application to 3D models remains challenging. Here, we present a step-by-step protocol for plating individual brain tumor neurospheres in the assay microplate to measure their bioenergetics. We provide actionable recommendations and highlight pitfalls to avoid. This approach enables exploration of dynamic metabolic changes in patient-derived brain tumor neurospheres following genetic or pharmacologic interventions. HIGHLIGHTS: Optimization of Seahorse assay protocol for single patient-derived brain tumor spheroidsStep-by-step instructions for successfully re-plating and transferring single brain tumor spheroidsImportance of microplate coating for the successful execution of Seahorse assaysImaging of the microplate before and after the assay allows for normalization of metabolic parameters based on neurosphere size.