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 |
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).