Extracellular Flux Analysis and 13C Stable-Isotope Tracing Reveals Metabolic Changes in LPS-Stimulated Macrophages

Importance of the Topic

Immune cell activation triggers profound metabolic remodeling that controls function and fate. Macrophages stimulated by lipopolysaccharide (LPS) switch from oxidative phosphorylation to aerobic glycolysis and synthesise antimicrobial metabolites such as itaconic acid. Understanding this rewiring at both phenotypic and pathway levels is essential for immunometabolism research, drug discovery, and industrial and clinical applications.

Study Overview

This application note demonstrates a combined workflow using Agilent Seahorse XFp real-time extracellular flux analysis and 13C stable-isotope tracing on an Agilent 6546 LC/Q-TOF with MassHunter VistaFlux software. RAW 264.7 murine macrophages were treated with 100 ng/mL LPS for four hours. Phenotypic changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured, and metabolic fluxes were traced after [U-13C6]-glucose labelling to reveal pathway-specific alterations.

Methodology and Instrumentation

Cell Culture and Phenotypic Assay

• RAW 264.7 cells cultured in DMEM with 10% FBS and seeded at 4 × 105 cells/mL in eight-well Seahorse miniplates.
• Cells pretreated ±100 ng/mL LPS for four hours, followed by measurement of basal and stressed OCR and ECAR on the Seahorse XFp Analyzer using the Cell Mito Stress Test kit.
• Kits and reagents: oligomycin, FCCP, rotenone/antimycin A, and TNFα ELISA for cytokine validation.

Stable-Isotope Tracing and Metabolite Extraction

• Cells labelled with 1:1 [U-13C6]-glucose/12C-glucose simultaneously with LPS treatment.
• Rapid quenching and extraction with cold acetonitrile/methanol/water buffer, followed by centrifugation and transfer to LC/MS vials.

LC/Q-TOF Analysis

• Chromatography: Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm) with alkaline ammonium acetate gradient at pH 9, 0.5 mL/min flow, 30 °C.
• Mass spectrometry: Agilent 6546 LC/Q-TOF in negative mode, m/z 40-1000, Jet Stream source, reference masses at m/z 68.9958 and 980.0164.

Data Processing

• Agilent MassHunter Profinder for isotopologue extraction and natural abundance correction.
• Target library generated by MassHunter Pathways to PCDL and retention times from in-house standards.
• Visualization of flux on pathways via Omix Premium module.
• Statistical analysis by unpaired two-tailed Student’s t-test, p < 0.05.

Key Results and Discussion

LPS treatment induced a 100-fold increase in secreted TNFα, confirming macrophage activation. Seahorse phenotype plots showed a marked shift toward a glycolytic profile (increased ECAR) under basal and stressed conditions. Stable-isotope tracing revealed nearly complete 13C labelling of pyruvate and lactate and substantial incorporation into glycolytic intermediates. In the TCA cycle, LPS enhanced 13C enrichment in cis-aconitate and itaconic acid while reducing labelling in the reductive branch, indicating a rerouting of carbon flux toward antimicrobial metabolite production.

Contributions and Practical Applications

This integrated workflow provides both macro-level bioenergetic phenotypes and micro-level pathway flux information in a single platform. It enables rapid mechanistic insights into immunometabolic regulation in response to stimuli or genetic perturbations. Applications span basic research, drug screening, metabolic biomarker discovery, and quality control in cell therapy and biotechnology.

Future Trends and Applications

Advances may include higher-throughput real-time flux analyzers, multiplexed isotope tracing with multiple labels (13C, 15N), AI-driven flux modeling, and integration with single-cell metabolomics. Such developments will support precision immunometabolism, personalized therapies, and dynamic monitoring of cell manufacturing processes.

Conclusion

The combination of Agilent Seahorse XF real-time bioenergetic profiling and 13C stable-isotope tracing on LC/Q-TOF with VistaFlux analysis offers a powerful strategy to dissect immune cell metabolic adaptations. This dual approach yields complementary phenotypic and flux data, enabling comprehensive understanding of metabolic reprogramming upon LPS-induced macrophage activation.

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