Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Summary

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Abstract

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

Introduction

Mitochondria play a major role in cell bioenergetics, which generates adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS). In addition to this, the role of mitochondria extends into the generation and detoxification of reactive oxygen species, cytoplasmic and mitochondrial matrix calcium regulation, cellular synthesis, catabolism, and the transport of metabolites within the cell¹. Mitochondrial respiration is essential in all cells, as their dysfunction can result in metabolic problems², including cardiovascular diseases³ and a wide variety of neurodegenerative diseases, such as age....

Protocol

Heparinized whole-blood samples were obtained from healthy human donors after obtaining informed consent, as approved by the Institutional Review Board of UConn Health in accordance with the Declaration of Helsinki. All animal experiments followed the UConn Health Institutional Animal Care and Use Committee (IACUC) guidelines, and approval for the use of rodents was obtained from the UConn Health IACUC according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Male C57BL/6 mice at 6 weeks of age were used in this study.

1. Preparation of the 96-well plate for the meta....

Results

Representative OCR dynamics are shown indicating the mitochondrial respiration changes in response to oligomycin, FCCP, and rotenone/antimycin A mixture of mouse neutrophils (Figure 3A), human neutrophils (Figure 3B), and undifferentiated and differentiated HL60 cells (Figure 3C). In all cells, oligomycin treatment decreases the OCR value by inhibiting the proton channel of ATP synthase; FCCP treatment restores the OCR value by incr.......

Discussion

The standard procedure that measures the mitochondrial respiration of neutrophils using the metabolic extracellular flux analyzer is limited by many factors, including cell number, cell growth, and viability. Each compound concentration varies among the type and source of cells in this assay. Oligomycin and rotenone/antimycin A are mostly used in a similar concentration among most cell types. However, as the FCCP-induced maximum respiratory rate varies among different cells, careful titration of FCCP is required to .......

Materials

Name	Company	Catalog Number	Comments
37 °C non-CO2 incubator	Precision	Economy Model 2EG	Instrument
Biorender			Software Application
Centrifuge	Eppendorf	Model 5810R	Instrument
Corning Cell-Tak Cell and Tissue Adhesive	Corning	102416-100	Reagent
EasySep Magnet	STEMCELL	18000	Magnet
EasySepMouse Neutrophil Enrichment kit	STEMCELL	19762A	Reagents
Graphpad Prism 9			Software Application
Human Serum Albumin Solution (25%)	GeminiBio	800-120	Reagents
Ketamine (VetaKet)	DAILYMED	NDC 59399-114-10	Anesthetic
PBS	Cytiva	SH30256.01	Reagents
Plate buckets	Eppendorf	UL155	Accessory
PolymorphPrep	PROGEN	1895 (previous 1114683)	polysaccharide solution
Purified mouse anti-human CD18 antibody	Biolegend	302102	Clone TS1/18
RPMI 1640 Medium	Gibco	11-875-093	Reagents
Seahorse metabolic extracellular flux analyzer	Agilent	XFe96	Instrument
Seahorse XF Cell Mito Stress Test Kit	Agilent	103015-100	mitochondrial stress test Kit
Swing-bucket rotor	Eppendorf	A-4-62	Rotor
Vactrap 2 Vacum Trap	Fox Lifesciences	3052101-FLS	Instrument
Wave			Software Application
XF 1.0 M Glucose Solution	Agilent	103577-100	Reagent
XF 100 mM Pyruvate Solution	Agilent	103578-100	Reagent
XF 200 mM Glutamine Solution	Agilent	103579-100	Reagent
XF DMEM medium	Agilent	103575-100	Reagent
XFe96 FluxPak	Agilent	102601-100	Material
Xylazine (AnaSed Injection)	DAILYMED	NDC 59399-110-20	Anesthetic