Begin with an adhesive-coated assay plate containing a monolayer of activated natural killer cells.
Position a pre-calibrated sensor cartridge containing fluorophore-embedded sensor probes, surrounded by multiple injection ports.
Load various mitochondrial respiration inhibitory compounds into distinct ports of the cartridge.
In an extracellular flux analyzer, sensors measure the cells' oxygen consumption rate, or OCR, with automatic compound injection.
In the first injection, oligomycin enters the cells' mitochondria and interacts with complex V of the electron transport chain, or ETC, blocking ATP synthesis and decreasing OCR.
The second injection introduces 2,4-dinitrophenol — a protonophore that shuttles protons across the mitochondrial membranes, causing proton leakage. To restore the membrane potential, oxygen consumption increases, maximizing the OCR.
Finally, with the third injection, rotenone and antimycin A bind to ETC's Complex I and III, respectively, stopping ETC and decreasing OCR to its lowest level.
The real-time OCR profile with inhibitors reveals the energy metabolism of natural killer cells.
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