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

Published: November 30, 2023

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…

Divulgazioni

The authors have nothing to disclose.

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

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Citazione di questo articolo
Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer. J. Vis. Exp. (Pending Publication), e21844, doi: (2023).

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