Seahorse xf96 cell culture microplate

For the quantification of ATP production rate from both glycolytic and mitochondrial pathways, Agilent Seahorse XF Real-Time ATP Rate Assay was performed according to the manufacturer’s instructions (Cat. 103592 Agilent Technologies) for the indicated conditions. For all adherent conditions, the day prior the assay 5000 cells/well, were seeded on a Seahorse XF96 cell culture microplate in DMEM 10% FBS, incubated at 37  ^oC and 10% CO₂. On the day of the experiment, media was changed for XF DMEM (pH=7.4) supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose. For floating conditions, 7500 A375P cells, previously grown under floating conditions for 24 h, were seeded on the Seahorse XF96 cell culture microplate in the XF media specified above. Both adherent and floating cells were then incubated at 37 °C without CO₂ for 1 h. During this incubation period, oligomycin and rotenone/antimycin A were prepared in Seahorse media to achieve final concentrations of 1.5 μM and 0.5 μM respectively when injected. The glycolytic and mitochondrial ATP production rate were calculated according to manufacturer instructions and normalized by protein content using Wave Desktop and Controller 2.6 software. Data are shown as XF ATP rate index, which is indicative of the mitoATP Production Rate divided by glycoATP Production Rate.

Cells were seeded in a Seahorse XF96 Cell Culture microplate (Agilent) and a regular 96-well plate. OCR and ECAR were acquired using the Seahorse XFe96 analyzer following manufacturer’s instruction for the Seahorse XF Cell Mito Stress Test Kit. Briefly, a sensor cartridge in Seahorse XF Calibrant was hydrated overnight at 37 °C in a non-CO₂ incubator and loaded with oligomycin (Port A), FCCP (Port B) and rotenone/antimycin-A (Port C). The medium in the Agilent Seahorse XF96 Cell Culture microplate was replaced with the Seahorse XF Base Medium supplemented with 10 mM of pyruvate, 2 mM of glutamine and 10 mM of glucose. The plate was then incubated for 1 h at 37 °C in the non-CO₂ incubator. Calibration of the cartridge was then performed (15–30 min) and the calibration plate was replaced with the cell culture microplate before running the experiment. OCR and ECAR data were normalized for cell numbers by using the Celigo Imaging Cytometer as described above.

The Cal27 and JHU022 cells (2 × 10⁴ cells/ well) were seeded into a 96 well-plate (Seahorse XF96 Cell Culture Microplates, Agilent) and treated with or without BME for 24–36 h. Cells were assessed for extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) to understand glycolysis rate (ECAR/ OCR) using the Seahorse XF analyser (Agilent) as described previously [21 (link)].