2.7. Seahorse XFp Protocol for Real-Time Metabolic Evaluation of U87MG Adherent Cells and GSCs Neurospheres

Experiments were performed in an XFp 8-well microplate using the Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). Briefly, GSCs were seeded at a density of 10,000 cells/well and allowed to grow for 72 h, in wells previously coated with 20 µL of Collagen Type IV at 20 µg/mL (C6745-1ML, Sigma Aldrich). U87MG cells were seeded at 6000 cells/well and allowed to grow for 24 h. Metabolic drugs were added to the treatment wells and fresh media was added to the control wells. After 72 h, the original media was carefully pipetted out of each well into a centrifuge tube without disturbing the attached cells; then, Seahorse XF DMEM medium, pH 7.4 (103575-100, Agilent) was used to wash, pipetted out and centrifuged with the original media at 1000 rpm for 5 min at 25 °C. After centrifugation, supernatant was aspirated from each tube, conserving only the cell pellet, resuspended in Seahorse medium and added back to respective wells.

We then followed the standard protocol for Standard XF Real-Time ATP Rate Assay, as described in the Seahorse XF Real-Time ATP Rate Assay User Guide (Kit 103592-100, Agilent). Seahorse XF technology measures two key parameters of cellular bioenergetics: oxygen consumption rate (OCR; an estimation of mitochondrial ATP) and extracellular acidification rate (ECAR; quantification of glycolytic activity through changes in pH by lactate production) [50,51]. Results were analyzed in Seahorse Wave software (version 2.6.1), with analysis of OCR and ECAR carried out using the Seahorse XF Real-Time ATP Rate Assay Report Generator (version 4.0.17). For normalization, total protein was quantified using an Invitrogen Qubit 3 Fluorometer ({"type":"entrez-protein","attrs":{"text":"Q33216","term_id":"75101668","term_text":"Q33216"}}Q33216, Invitrogen).