Protocol for real-time assessment of mitochondrial and glycolytic ATP production in patient-derived glioma stem-like cells

Summary: Glioma cells switch between energetic pathways to adapt and resist therapies. We present a protocol for measuring mitochondrial and glycolytic ATP rates in patient-derived glioma stem-like cells using a Seahorse XF ATP rate assay. We describe steps for growing 3D glioma stem-like cells, attaching cells to the assay plate, preparing drugs, and running the ATP rate assay. We also detail procedures for imaging viable cell numbers and normalization, with tips to overcome pitfalls in Agilent Seahorse assays. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.