Metabolism is more than just ATP production—it serves as the backbone of cellular function, directly or indirectly driving key processes such as cell growth, differentiation, and functional specialization. How cells shift their metabolic preference between glycolysis and mitochondrial respiration can play a causal role in shaping functional demands and signaling states, including activation and quiescence.
Real-time measurement of cellular metabolism, uniquely enabled by Agilent’s Seahorse XF technology, simultaneously captures a cell’s oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which reflect mitochondrial respiration and glycolysis, respectively. This functional approach is widely recognized as the gold standard for measuring metabolism in living cells.
Although such experiments have traditionally been performed with 2-dimensional (2D) cell cultures, metabolic characterization using advanced 3D model systems, such as organoids and precision tissue slices, promises more physiologically relevant insights. A novel, next-generation innovation improves upon the original Seahorse XF Technology, enabling dynamic metabolic analysis of 3D study models for the first time.
Agilent Seahorse XF analyzers are designed to measure the rates of oxygen consumption and proton efflux in the medium immediately surrounding living cells in a microplate. To achieve this, they use a biosensor cartridge containing two polymer-embedded fluorophores, one of which is quenched by oxygen and the other by protons.
During a measurement cycle, the XF sensor cartridge is lowered into the microplate, creating a semi-sealed, transient microchamber in each well. The fluorophores are then excited by light and changes in fluorescence emissions are monitored over a period of 2–5 minutes.
The assay can be customized using drug injection ports within the XF sensor cartridge, which allow for the addition of inhibitors, activators, substrates, or other compounds of interest. Once the measurement period is complete, the sensor cartridge lifts up to re-establish initial conditions before the next measurement cycle begins.
Importantly, Agilent Seahorse XF technology enables monitoring of a cell’s functional bioenergetic changes in real time without the need for invasive methods such as dyes or labels that can alter cell function.
Figure 1. Principles of Agilent Seahorse XF technology
To enable real-time metabolic analysis of advanced 3D study models, Agilent has developed a novel Seahorse XF analyzer, the Seahorse XF Flex. This new innovation works with specific solutions to facilitate advanced 3D studies, while remaining fully compatible with traditional 2D models. Using the newly launched Seahorse XF Flex instrument in combination with specialized 3D capture microplates and an optimized 3D Mito Stress Test Kit, a tissue workflow protocol was developed and validated with ex vivo rat brain samples. Notably, because tissue samples require higher compound doses and longer penetration times than 2D cultures, optimization was required for different compound concentrations and numbers of measurement cycles.
The customized XF workflow incorporates a standardized method for preparing tissue slices. Briefly, freshly harvested whole rat brains are embedded in agarose and sliced into 200 µm coronal sections using a microtome. Tissue punches (1–2 mm) are then prepared using stainless steel biopsy tools and carefully transferred into the microplate wells. Here, samples are held in place with 3D capture rings, which have a large open mesh to allow for good reperfusion and recovery during analysis. By implementing measures that ensure consistent slice size, shape, and placement, as well as preserve tissue viability, assay variability is minimized for more consistent experimental results.
To streamline the implementation of Seahorse XF technology for studying advanced 3D cellular models, the customized workflow is designed for integration with Agilent Wave Pro software and Agilent Seahorse analytics tools. In addition, the optimized XF 3D Mito Stress Test Kit includes standard modulators of mitochondrial respiration, with concentrations tailored for 3D models. Together with the standardized protocol, these measures support efficient assay setup, compound titration, and data acquisition for both dose-response and screening applications.
The heterogeneity of advanced 3D model systems makes normalization crucial. The customized workflow supports three complementary normalization strategies for increased flexibility in experimental design:
By using one or more of these methods, researchers can improve cross-sample comparisons and increase confidence in data interpretation.
Although validated only with precision rat brain slices to date, the customized workflow for real-time metabolic analysis of 3D study models is adaptable to other organisms and tissue types, as well as a broad range of disease models. This translationally relevant platform bridges the gap between traditional cell cultures and in vivo studies for drug development and mechanistic research, providing researchers with new opportunities for discovery.
Learn more about Agilent Seahorse XF technology here