Xf96 v3 ps cell culture microplate

Cell suspensions of acutely isolated astrocytes by immunomagnetic purification were seeded in 12‐well plates (Corning, #351143) at a density of 130,000 cells/well in DMEM with glucose, GlutaMAX(TM) and pyruvate (Thermo Fisher Scientific, #31966) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. The medium was exchanged every second day. On day 10, the cells were transferred in XF96 V3 PS cell culture Microplates (Agilent Technologies) coated with 0.01% Poly‐d‐Lysin (Thermo Fisher Scientific) at a density of 40,000 cells/well in the same culture medium and the Seahorse assay took place 5 days later. On the day of measurement, the cells were washed with XF assay medium (Agilent Technologies) containing 1 mM sodium pyruvate and 2 mM glutamine and incubated in fresh XF medium for 1 h under CO₂‐free conditions. Thereafter, oxygen consumption and extracellular acidification rates (OCR and ECAR) were measured simultaneously using a Seahorse XFe96 Flux Analyzer (Agilent Technologies). The following injections and final concentrations were used: glucose (1 μM) oligomycin (2 μM), FCCP (2 μM) and antimycin A (0.5 μM)/rotenone (0.5 μM). Data were normalized to total DNA per well as a surrogate for cell number per well.

5 × 10⁴ PC128 or 9 × 10⁴ U251 cells were seeded on glass cover inserts placed in 6-well plates and allowed to attach overnight. Then, the cover slips were transferred to TTFields ceramic dishes and treated for 24 h. Afterwards, the cells were enzymatically detached and 1 × 10⁴ cells/well were seeded on XF96 V3 PS cell culture microplates (Agilent Technologies Inc., Wilmington, DE, USA) and allowed to attach for 24 h followed by one wash with XF assay medium containing 5 mM glucose (pH adjusted to 7.5). Afterwards, the mito stress test kit (Agilent Technologies Inc.) was used as described by the manufacturer applying serial injections of oligomycin at a final concentration of 2 µM, FCCP at a final concentration of 2 µM and rotenone/antimycin A at a final concentration of 0.5 µM. All analyses were performed on an Agilent Seahorse XFe96 analyzer.

1 × 10⁴ cells were seeded on XF96 V3 PS cell culture microplates (Agilent Technologies Inc., Wilmington, DE, U.S.A.). After 24 h, cells were subjected to the indicated treatments for 24 h followed by washes with XF assay medium containing 5 mM glucose (pH adjusted to 7.5). Afterwards the mito stress test kit (Agilent Technologies Inc., Wilmington, DE, U.S.A.) was used as described by the manufacturer applying serial injections of oligomycin at a final concentration of 2 µM, FCCP at a final concentration of 2 µM and rotenone/antimycin A at a final concentration of 0.5 µM. All analyses were performed on an Agilent Seahorse XFe96 analyzer.

1 × 10⁴ cells were seeded on XF96 V3 PS cell culture microplates (Agilent Technologies Inc., Wilmington, DE, United States). After 24 h cells subjected to the indicated treatments for 24 h followed by washes with XF assay medium containing 5 mM glucose (pH adjusted to 7.5). Afterwards the mito stress test kit (Agilent Technologies Inc., Wilmington, DE, United States) was used as described by the manufacturer applying serial injections of oligomycin at a final concentration of 2 μM, FCCP at a final concentration of 2 µM and rotenone/antimycin A at a final concentration of 0.5 µM. All analyses were performed on an Agilent Seahorse XFe96 analyzer.

Single cell suspensions were prepared from macro-dissected tumors using gentleMACS Octo Dissociator (Miltenyi Biotec). Macro-dissected tumors were weighted and processed into DMEM, no glucose (11966025, ThermoFisher Scientific). Ten or five technical replicates per tumor (5 mg of tumor per replicate) were plated in a XF96 V3-PS cell culture microplate (Seahorse Bioscience, North Billerica) and the rest of cell suspensions were saved for real-time PCR. Cells were incubated during 2 hr in DMEM, no glucose at 37°C. Extracellular acidification rates (ECAR) were then measured using XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA). ECAR measurements were performed at 37°C, with measurement cycles broken down into 2 min of mixing, 2 min of waiting and 3 min of data acquisition. During Seahorse assay, 10 mM of glucose was added, followed by 50 mM of 2-deoxyglucose (2-DG). ECAR measurements were reported as normalized to protein concentration. After Seahorse analysis, proteins were extracted from the cells for western blot analyses.