Seahorse xfe xf analyzer

To estimate the rate of glycolysis and mitochondrial respiration, a Seahorse XFe/XF Analyzer (Agilent, Santa Clara, CA, USA) was used according to the manufacturer’s protocol. Briefly, 4 × 10⁴ cells/well were seeded in culture medium and the sensor cartridge was hydrated in Seahorse XF Calibrant buffer at 37 °C in incubator in absence of CO₂ for 24 h. Then, culture medium was removed and replaced by Seahorse assay media. For the mitochondrial respiration test, the medium (pH 7.4) contained 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. The mitostress assay was performed by sequentially injecting 1 μM oligomycin, 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 μM rotenone/0.5 μM antimycin A. The cell number adjustment/normalization was performed using BCA.

The cell oxygen consumption rate (OCR) was measured using an Agilent Seahorse XF Cell Mitochondrial Stress Test kit (103015-100) to characterize the key parameters of mitochondrial function. HK-2 cells in the culture plate were treated with 10 μg/mL of NMs and cultured for 24 h. Subsequently, the user guide for the experiment was followed. Using a Seahorse XFe/XF analyzer (Agilent, Santa Clara, CA, USA), the data were analyzed using the Seahorse XF Mitochondrial Stress Test Report Generator. At least three biological replicates were examined for each experiment.