Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines

Abstract

Metabolic networks are fundamental to cellular processes, driving energy production, biosynthesis, redox regulation, and cellular signaling. Recent advancements in metabolic research tools have provided unprecedented insights into cellular metabolism. Among these tools, the extracellular flux analyzer stands out for its real-time measurement of key metabolic parameters: glycolysis, mitochondrial respiration, and fatty acid oxidation, leading to its widespread use. This review provides a comprehensive summary of the basic principles and workflow of the extracellular flux assay (the Seahorse assay) and its diverse applications. We highlight the assay's versatility across various biological models, including cancer cells, immunocytes, Caenorhabditis elegans, tissues, isolated mitochondria, and three-dimensional structures such as organoids, and summarize key considerations for using extracellular flux assay in these models. Additionally, we discuss the limitations of the Seahorse assay and propose future directions for its development. This review aims to enhance the understanding of extracellular flux assay and its significance in biological studies.

Keywords: Electron transport chain; Extracellular flux assay; Glycolysis; Metabolism; Mitochondria; Seahorse assay.

Fig. 1

ECAR and OCR as proxies of glycolysis and mitochondrial respiration in Seahorse assay. Glucose is metabolized to pyruvate through glycolysis. The pyruvate can be converted to lactate and protons (H+), leading to extracellular acidification. Fatty acyl-CoA is converted to acyl-carnitine by carnitine palmitoyltransferase-1 (CPT-1) to enter the mitochondria, where it is converted back to fatty acyl-CoA and undergoes FAO to produce acetyl-CoA. The acetyl-CoA generated from glycolysis and FAO enters the TCA cycle, leading to ATP production through oxidative phosphorylation, which consumes oxygen in mitochondria. Thus, the quantification of ECAR or OCR represents the capacity of glycolytic and mitochondrial respiration, respectively. (a) General patterns of the graph during testing the glycolytic functions using the Seahorse assay. (b) General patterns of the graph obtained during the Mitostress test for in-depth analysis of mitochondrial function. (c) General results for the measurement of FAO using the Seahorse assay.

Fig. 2

The workflow of Seahorse assay. The Seahorse assay process includes steps from optimization of conditions to data interpretation. The optimization step involves drug optimization (eg, oligomycin, FCCP) and optimization of sample quantity (eg, cell seeding number, quantity of mitochondria). Generally, 1 day before the assay, the cartridge is hydrated, and cells are seeded on the assay plate. On the day of the assay, the media is replaced with the assay media, drugs are prepared and loaded into the ports, and the program is run once the cartridge calibration is finished. When the program ends, cells or materials are subjected to further quantification for normalization. After the data are normalized, it is interpreted.

Fig. 3

Targets of the drugs utilized in the Seahorse assay. In the measurement of OCR, oligomycin, FCCP, and rotenone/antimycin A are sequentially utilized. Oligomycin, an inhibitor of ATP synthase, allows the measurement of ATP-linked respiration. FCCP, a mitochondrial uncoupler, disrupts the proton gradient across the mitochondrial membrane, inducing maximal respiration. Rotenone and antimycin A are typically used together as inhibitors of complexes I and III, respectively. Their action results in the complete shutdown of the electron transport chain. In the FAO measurement, palmitate serves as the substrate for FAO, while etomoxir acts as an inhibitor that prevents FAO by blocking CPT-1. In the measurement of ECAR, glucose, oligomycin, and 2-DG are utilized. Glucose transporters (GLUTs) facilitates the transport of both glucose and 2-deoxyglucose (2-DG) across the plasma membrane. Glucose is the substrate for glycolysis. Oligomycin in ECAR measurements blocks the electron transport chain, maximizing glycolysis. 2-DG, a glucose derivative and competitive inhibitor of glycolysis, is phosphorylated to 2-deoxyglucose-6-phosphate (2-DG-6-P), which terminates glycolysis immediately by binding to hexokinase.

Fig. 4

Applications of Seahorse assay. The Seahorse assay is applicable in various models, from cancer cells to model organisms such as C. elegans, for in-depth analysis of mitochondrial function and glycolysis in metabolism.