Seahorse xf analyzer

Cells were incubated with the XF base medium without phenol red, which was supplemented with 5.0 mM HEPES (Thermo Fisher Scientific), 10 mM glucose, 2 mM glutamine (Caisson Labs), and 1 mM pyruvate (Invitrogen) for 1 hour in a non-CO₂ incubator at 37°C. Meanwhile, a prehydrated Seahorse XF Sensor Cartridge was loaded with rotenone/antimycin A (50 μM) and 2-DG (500 mM) into port A and port B, respectively. The Seahorse XF Analyzer was calibrated, and the assay was performed using the Seahorse XF GRA protocol as suggested by the manufacturer. Proton efflux rate was recorded, which was normalized to numbers of living cells in representative wells.

Mitochondrial utilization of fatty acids was measured by the Seahorse XF Analyzer (Seahorse Bioscience, North Billerica, MA) according to the instructions for the Seahorse XF Mito Fuel Flex Test Assay [10 (link)]. Briefly, oxygen consumption rate (OCR) was measured after sequential injection of these inhibitors: etomoxir, a specific Cpt1 inhibitor, 4 μM; UK5099, a mitochondrial pyruvate carrier inhibitor, 2 μM; BPTES, a selective glutaminase inhibitor, 3 μM and after optimization. The FAO measures reliance on fatty acid utilization to maintain baseline respiration, which was calculated using the formula: dependency (%) = (baseline OCR -target OCR)/(baseline OCR- all inhibitors OCR) × 100.

The real-time oxygen consumption rate (OCR) of SK-NBE clones (CTRL and ΔNAGLU) was measured at 37°C using a Seahorse XF Analyzer (Seahorse Bioscience, North Billerica, MA, USA). SK-NBE clones were plated into specific cell culture microplates (Agilent, Santa Clara, CA, USA) at the concentration of 3x10⁴ cells/well, and cultured for 12 h in DMEM, 10% FBS. OCR was measured in XF media (non-buffered DMEM medium, containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) under basal conditions and after sequential addition of 1.5 μM oligomycin, 2 μM FCCP, and rotenone + antimycin (0.5 μM all) (all from Agilent). Indices of mitochondrial respiratory function were calculated from the OCR profile: basal OCR (before addition of oligomycin), maximal respiration (calculated as the difference between FCCP rate and antimycin + rotenone rate), spare respiratory capacity (calculated as the difference of FCCP-induced OCR and basal OCR), ATP production (calculated as difference between basal OCR and oligomycin-induced OCR) and proton leak (calculated as the difference between the minimum rate measurement after oligomycin injection and non-mitochondrial respiration). Reported data were the mean values ± SEM of three measurements deriving from two independent experiments.

Using the Seahorse XF analyzer (Seahorse Bioscience, North Billerica, MA), in real time, mitochondrial oxidative phosphorylation and glycolytic flux can be analyzed by measuring OCR and ECAR of cells as they respond to substrates and metabolic inhibiting agents according to manufacturer’s instructions [34 (link)]. ATP synthase inhibitor oligomycin (a complex V blocker) is added to inhibit coupled respiration. FCCP (a mitochondrial uncoupler) is added to collapse mitochondrial membrane potential (Δψm). Rotenone (an inhibitor of complex I of the electron transport chain) and antimycin A (an inhibitor of complex III of the electron transport chain) were add to block mitochondrial respiration completely. To analyze real-time glycolytic rates, Seahorse XF glycolytic rate assay utilizes both (extracellular acidification rate) ECAR and OCR measurements to evaluate the glycolytic proton efflux rate (glycoPER) of the cells, in which the cells were incubated in glucose-free media followed by the addition of rotenone, antimycin A, and finally 2-deoxyglucose (2-DG, glycolysis inhibitor). OCR and ECAR were described as absolute rates (pmoles/min for OCR and mpH/min for ECAR) and normalized against cell counts as a percentage of the baseline oxygen consumption.

HCT116 cells with a density of 6 × 10⁴ cells ml⁻¹ of were seeded on a XF24-plate containing blank controls. The Seahorse XF analyzer was used as indicated by the manufacturer (Seahorse Bioscience). Before the measurements, medium was replaced with 500 μL of Seahorse XF base medium (200 mM L-glutamine, 1 mM pyruvate, 2.5 M glucose, pH 7.4) at 37°C without CO₂ for 1 h. Oxygen consumption rate (OCR) values were measured by XF24 Extracellular Flux Analyzer. The assessment of Mitochondrial activity was performed using a Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience). All experiments were performed in triplicates and were repeated three times.