Extracellular flow analyzer xf96

Then measurement of extracelluler acidification rate (ECAR) and oxygen consumption rate (OCR) were detected by XF96 Extracellular Flow analyzer (Seahorse Bioscience, Billerica, Ma, USA) in real time. Briefly, 1 × 10⁴ cells/well SKOV3 cells were cultured in custom XF96 microplates. After cell attaching well (~12 h), the media was replaced with 80 μl of RPMI-1640 media containing indicated concentration of TPL for 24 h. Before measurement, non-buffer XF assay medium was preheated to 37 °C, and cells were washed with the assay medium twice. Immersed in 180 μl non-buffered medium and incubated in the 37 °C, and non-CO₂ incubator for 1 h. All experiments were performed at 37 °C. In order to eliminate the effect of the cell proliferation rate, determined the concentrations of protein in each well after finishing the assay and then normalized either OCR or ECAR by dividing the concentration. Cellular viabilities that were determined after assay were nearly indistinguishable regardless of 24 h TPL exposure or not. For measurement of ECAR, glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM) were added at a specified point in time. For measurement of OCR, oligomycin (1 μM), FCCP (0.5 μM), and rotenone-antimycin A (0.5 μM) were added at a specified point in time. The bracketed concentrations of drugs are the final concentrations.