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

• Published in: STAR protocols 2024-09, Vol.5 (3), p.103159, Article 103159
• Main Authors: Sharma, Pratibha, Puduvalli, Vinay K.
• Format: Article
• Language: English
• Subjects: Cancer; Cell Biology; Cell culture; Cell-based Assays; Health Sciences; Metabolism; Neuroscience; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2024.103159

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. [Display omitted] •Simultaneous measurement of mitochondrial and glycolytic ATP production•Use of floating, patient-derived glioma stem-like cells•Detailed and optimized protocol for profiling bioenergetics for non-adherent cells•Image-based normalization to overcome the variations that arise from other methods Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.