Xf24 cell tak

Experiments were carried out on Seahorse XF24 bioanalyzer (Agilent, USA). The oxygen consumption rate (OCR) was measured as described following the Mito Stress Assay Kit (Cat. 103674-100). Briefly, a total of 5 × 10⁴ cells were seeded per well onto XF24 Cell-Tak (BD Bioscience, Cat. 354240), and cultured in growth medium for 12 h. And measurement was collected following incubation with Seahorse XF DMEM media supplemented with glutamine, sodium pyruvate, and glucose. And the 1.5 μmol/L ATP synthase inhibitor of complex V (oligomycin), 1.0 μmol/L mitochondrial uncoupler (FCCP) and 0.5 μmol/L ETC inhibitors of complexes I and III (rotenone with antimycin A) were injected from the XF24 ports as indicated to examine the basal respiration rate and maximum respiration rate. The analysis was performed using Seahorse Wave software (v. 2.6.3).

The assays were performed with an Agilent Seahorse XF24 bioanalyzer (Agilent, Santa Clara, USA). Briefly, LX2 cells were isolated and spun onto XF24 Cell-Tak (# 354240 BD Bioscience, New Jersey, USA) coated plates in Seahorse XF RPMI 1640 media supplemented with glutamine, sodium pyruvate, and glucose. For immediate metabolic response, cells were treated with 1.0 µM concentrations of oligomycin (ATP synthase inhibitor of complex V), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, electron transport chain uncoupler), and rotenone with antimycin A (which are electron transport chain complex I and III uncouplers, respectively) throughout the analysis. Mitochondrial oxidative phosphorylation was measured by the oxygen consumption rate (OCR). All assays were performed in triplicate, and repeats were performed for this experiment.