Seahorse xf24 extracellular flux analyzer

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cells and mitochondria were determined using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience Inc., Chicopee, MA, USA). For comparison between experiments, the data are expressed as OCR of pmol/min/1 × 10⁵ cells and ECAR of mpH/min/1 × 10⁵ cells. In the beginning, 1 × 10⁵ cells were seeded on Seahorse XF24 Cell Culture Microplates. After overnight incubation, cells were treated with 0, 0.01, 0.1, 1, and 10 μM BA6 for 24 h. After washing the cells with 0.5 ml of DMEM without sodium bicarbonate, pH = 7.4, and then, 675 μl of DMEM was added to each well for further examination. The base OCR was measured four times, plotted as a function of the cells under basal conditions, and then, 1 μM oligomycin, 0.25 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and 1 μM rotenone were added sequentially as indicated (Figure 1(a)). At the end of recording, cells were collected and counted by using a trypan blue exclusion assay, and then, the OCR and ECAR values were calculated after normalization with the number of cells.

The ECAR was analyzed using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). In brief, 2 × 10⁵ cells were seeded into each well of a Seahorse XF24 cell culture microplate. On the next day, the cells were treated with/without CLQ at indicated doses for 24 h, and GW9662 was added when necessary. Then, cells were switched to a basal glucose-free medium for 1 h prior to measurement. A default standard glycolysis stress-test program was performed. During measurement, glucose (final concentration 10 mM), oligomycin (final concentration 1 μM) and 2-DG (final concentration 100 mM) were sequentially injected into each well at the indicated time points. Data were derived from Seahorse XF24 Wave software and displayed in pmol/minute.