Seahorsexf basic medium

Cancer cells under a hypoxic environment mainly depend on glycolytic pathways as a source of cell energy. Therefore, extracellular acidification rate (ECAR) was measured by a Seahorse XF24 Extracellular Flux Assay Kits (Agilent technologies, Q29216) and Seahorse XF24 Extracellular Flux Analyzers (Agilent technologies) in order to detect glycolysis. HCC827 cells were planted in a Seahorse XF24 Cell Culture Microplates (Agilent Technologies, 28816) at a density of 1 × 10⁵ cells/well. 1 mL hydration fluid was added into the lower layer of the XF24 Extracellular Flux Assay Kit and hydrated overnight in a CO₂-free incubator at 37°C. Meanwhile, the drug and Seahorse XF basic medium (Agilent technologies, 102353) were prepared, of which the pH value was adjusted to 7.4. The medium was put into 37°C water bath for 1 h before washing the cells twice with it. Then each well was replenished by 450 μl Seahorse XF basic medium. Culture cells for another 1 h at 37°C. Different concentrates of CHE were diluted, and 75 μl solution was added into the upper layer of XF24 Extracellular Flux Assay Kits for measurements. After 30 min, the lower layer of XF24 Extracellular Flux Assay Kits was removed and replaced by the cell plate for detecting.

The glycolysis stress test was conducted by using Seahorse XF Glycolysis Stress Test Kit (#103,020–100, Agilent Technologies, USA). SeahorseXF basic medium (#103,334–100, Agilent Technologies, USA) was added with 2 mmol glutamine as the detection solution. Oligomycin, glucose and 2-DG were properly prepared into a working solution, which was correspondingly added into the dosing hole on the probe board. The cell growth medium was replaced by a warm detection solution, and the cell culture microplate was placed in a CO₂-free incubator at 37℃ for an hour. SeaHorseXF24 (Agilent Technologies, USA) was then run on the computer and the data were analyzed.

The oxygen consumption rate (OCR) was conducted using Seahorse XF Cell Mito Stress Test Kit (#103,015–100, Agilent Technologies, USA). SeahorseXF basic medium (#103,575–100, Agilent Technologies, USA) was added with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose as the detection solution. Oligomycin, FCCP and Rot/AA were prepared correctly into a working solution, which was correspondingly added into the dosing hole on the probe board. The cell growth medium was replaced by a warm detection solution, and the cell culture microplate was placed in a CO₂-free incubator at 37℃ for an hour. SeaHorseXF24 (Agilent Technologies, USA) was then run on the computer and the data were analyzed.