Agilent seahorse xf extracellular flux analyzer

FAO and OCR were assessed using an Agilent Seahorse XF extracellular flux analyzer (Agilent Technologies) according to a previously described method.^[46 (link),
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^] For FAO analysis, podocytes were plated on Seahorse assay plates and treated with Seahorse XF assay medium containing DMEM with 5.5 mm glucose and 0.5 mm carnitine according to the manufacturer's instructions from Seahorse Bioscience. A specific CPT‐1 inhibitor Etomoxir (ETO, 250 µm) was injected to evaluate the rate of FAO, and then a blocker of glycolysis and pyruvate oxidation 2‐deoxyglucose (2‐DG, 50 mm) was injected to assess the glucose utilization. For the OCR analysis, podocytes were seeded in Seahorse assay plates and treated with Seahorse XF assay medium. After obtaining the baseline OCR, the measurements were obtained after sequential injection of 5 µm oligomycin, 1.5 µm FCCP, and 5 µm rotenone.

Cell mitochondrial respiration and glycolytic activity were measured using “Agilent Seahorse XF Cell Energy Phenotype Test Kit” (Agilent Dako, Santa Clara, CA, USA) according to the manufacturer's instructions. Plate wells were coated with poly-D-lysine 24 hours before measurement. NB4 cells were seeded at 3 x 10⁴ cells/well, centrifuged for 1 min at 300xg at room temperature, and incubated for 30 min at 37°C without CO₂. After incubation, cell metabolic phenotype was measured on the Agilent Seahorse XF Extracellular Flux Analyzer (Agilent). Determined oxygen consumption rate (OCR) demonstrates mitochondrial respiration and extracellular acidification rate (ECAR), rate of glycolysis of the cells.