Protocol for Seahorse 3D Mito Stress assay in patient-derived atypical teratoid rhabdoid tumor CHLA-05-ATRT single neurospheres

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 protocol for plating individual brain tumor neurospheres in the assay microplate to measure their bioenergetics. We describe steps for neurosphere culturing, cartridge hydration, microplate coating, and neurosphere transfer. We then detail procedures for incubation and Seahorse assay, followed by normalization and analysis. This approach enables exploration of dynamic metabolic changes in patient-derived brain tumor neurospheres at basal conditions or following genetic or pharmacologic interventions.