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

Abstract

Source: Traba, J. et al., Analysis of Human Natural Killer Cell Metabolism. 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.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Reagent preparation Reagents for the isolation of natural killer (NK) cells NOTE: Prepare these reagents in a cell culture hood. 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…

Materials

2-Deoxy-D-glucose (2-DG)	MilliporeSigma	D8375-5G	Glycolyisis stress test injector compound
2,4-Dinotrophenol (2,4-DNP)	MilliporeSigma	D198501	ETC uncoupler / mitochondrial stress test injector compound
96 Well Cell Culture Plate/ Round bottom with Lid	Costar	3799	NK cell culture
Antimycin A	MilliporeSigma	A8674	Complex III inhibitor / glycolysis and mitochondrial stress test injector compound
BD FACSDIVA Software	BD Biosciences		Flow data acquisition
BD LSR Fortessa	BD Biosciences		Flow data acquisition
Cell-Tak	Corning	354240	Cell adhesive
EasySep Human CD3 Positive Selection Kit II	Stemcell technologies	17851	NK cell isolation from PBMCs
EasySep Human NK cell Enrichment Kit	Stemcell technologies	19055	NK cell isolation from PBMCs
EasySep Magnet	Stemcell technologies	18001	NK cell isolation from PBMCs
EDTA 0.5 M, pH 8	Quality Biological	10128-446	NK sell separation buffer
FACS tubes	Falcon-Fisher Scientific	352235	Flow cytometry experiment
Falcon 50 ml Conical tubes	Falcon-Fisher Scientific	14-432-22	NK cell separation
Fetal Calf Serum (FCS)	Gibco	10437-028	NK cell separation buffer
FlowJo Software	BD Biosciences		Flow data analysis
Glucose	MilliporeSigma	G8270	Component of mitochondrial stress test medium. Glycolysis stress test injector compound
Human IL-15	Peprotech	200-15-50ug	NK cell stimulation
Human serum (HS)	Valley Biomedical	9C0539	NK cell culture medium supplement
IMDM	Gibco	12440053	NK cell culture medium
L-Glutamine (200 mM)	ThermoFisher Scientific	25030-081	Component of stress test media
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit	ThermoFisher Scientific	L34965	Viability dye for flow cytometry staining
LSM	mpbio	50494X	PBMCs separation from human blood
Mouse anti-human CD3 BV711	BD Biosciences	563725	T cell flow cytometry staining
Mouse anti-human CD56 PE	BD Pharmingen	555516	NK flow cytometry staining
Mouse anti-human NKp46 PE	BD Pharmingen	557991	NK flow cytometry staining
Oligomycin	MilliporeSigma	75351	Complex V inhibitor / mitochondrial stress test injector compound
PBS pH 7.4	Gibco	10010-023	NK cell separation buffer
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23225	For determination of protein concentration
Rotenone	MilliporeSigma	R8875	Complex II inhibitor / glycolysis and mitochondrial stress test injector compound
Seahorse Wave Controller Software	Agilent		Controller for the Seahorse XFe96 Analyzer
Seahorse Wave Desktop Software	Agilent		For data analysis
Seahorse XF Base Medium	Agilent	102353-100	Extracellular Flux assay base medium
Seahorse XFe96 Analyzer	Agilent		Extracellular Flux Analyzer
Seahorse XFe96 FluxPak	Agilent	102416-100	Includes 20 XF96 cell culture plates, 18 XFe96 sensor cartridges, loading guides for transferring compounds to the assay cartridge, and 1 bottle of calibrant solution (500 ml).
Sodium bicarbonate	MilliporeSigma	S5761	To prepare the Cell-Tak solution
Sodium pyruvate (100 mM)	ThermoFisher Scientific	11360-070	Component of mitochondrial stress test medium