Immunology JoVE (Open Access) Citable · DOI

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Name: Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis
Uploaded: 2026-06-17
Duration: 7 min 45 s
Description: Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

DOI: 10.3791/53424-v

What you'll learn

✓ Polarize bone marrow-derived macrophages into M1 and M2 phenotypes
✓ Measure glycolytic and mitochondrial function using extracellular flux analysis
✓ Interpret metabolic differences between macrophage polarization states

Protocol

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

Difficulty

intermediate

Total time

~7-10 days (including bone marrow isolation, macrophage differentiation, polarization, and assay execution)

Model organism

Mouse bone marrow-derived macrophages

Biosafety

BSL-1

Steps

Polarize bone marrow-derived macrophages into M1 and M2

Culture isolated mouse bone marrow cells and treat with appropriate cytokine stimuli (IFN-γ + LPS for M1; IL-4 for M2) to induce distinct macrophage polarization states. Control M0 macrophages serve as baseline.

▶ 00:59

Prepare samples and extracellular flux assay plate

Harvest polarized macrophages, normalize cell numbers, seed them into assay plates, and prepare the instrument according to manufacturer protocols for real-time metabolic measurement.

▶ 02:17

Execute bioenergetic characterization of polarized macrophages

Run the extracellular flux assay to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in response to sequential injection of metabolic modulators, characterizing glycolytic and mitochondrial function.

▶ 03:46

Analyze metabolic characteristics across macrophage phenotypes

Compare obtained OCR and ECAR values across M0, M1, and M2 macrophages to identify metabolic signatures associated with each polarization state and phenotype.

▶ 05:17

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