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bioRxiv · 10.64898/2025.12.03.691986

Protocol for Seahorse 3D Mito Stress assay in patient-derived pediatric brain tumor single neurospheres

Tocci, S.·Tsai, J. W.
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Abstract

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/691986v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@25cb34org.highwire.dtl.DTLVardef@13ff857org.highwire.dtl.DTLVardef@c91c7forg.highwire.dtl.DTLVardef@16dfa27_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIOptimization of Seahorse assay protocol for single patient-derived brain tumor spheroids C_LIO_LIStep-by-step instructions for successfully re-plating and transferring single brain tumor spheroids C_LIO_LIImportance of microplate coating for the successful execution of Seahorse assays C_LIO_LIImaging of the microplate before and after the assay allows for normalization of metabolic parameters based on neurosphere size C_LI

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Tocci, S., Tsai, J. W.. 2025-12-05. Protocol for Seahorse 3D Mito Stress assay in patient-derived pediatric brain tumor single neurospheres. https://doi.org/10.64898/2025.12.03.691986

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