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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Reagent preparation
<ol>
	<li>Reagents for the isolation of natural killer (NK) cells 
	NOTE: Prepare these reagents in a cell culture hood.
	<ol>
		<li>Prepare NK separation buffer: Supplement phosphate-buffered saline, PBS (pH 7.4) with 1 mM ethylenediaminetetraacetic acid (EDTA) and 2% fetal calf serum (FCS) that h...

real-time monitoring of energy metabolism in human nk cells using an extracellular flux analyzer

Source: Traba, J. et al., <a href="https://app.jove.com/v/61466">Analysis of Human Natural Killer Cell Metabolism.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates the method to analyze energy metabolism in activated human NK cells using an extracellular flux analyzer. Different inhibitors of the electron transport chain complexes impact ATP synthesis, which is measured through the oxygen consumption rate or OCR, providing insights into cellular metabolism and bioenergetics.

Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Reagent preparation

	Reagents ...

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&#39; oxygen consumption rate, or OCR, with automatic compound injection.
In the first injection, oligomycin enters the cells&#39; mitochondria and interacts with complex V&#160; of the electron transport chain, or ETC, blocking ATP&#160; synthesis and decreasing OCR.
The second injection introduces 2,4-dinitrophenol &#8212; 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&#160; bind to ETC&#39;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.