Background

Seahorse metabolic assays have been widely adopted across numerous fields, spanning immunology, cancer, genetics, neuroscience, pharmacology, and others, to provide real-time measurements of cellular metabolism in live cells. Seahorse assays offer a standardized method to interrogate glycolysis and mitochondrial function through assessments of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. Extracellular acidification rate is calculated from changes in pH over time that reflect differences in proton efflux to act as a surrogate for glycolysis measurements. Oxygen consumption rate is determined through measurements of dissolved oxygen levels and reflects mitochondrial respiration. pH changes and oxygen levels are measured by sensor probes within the Seahorse Bioanalyzer every few seconds directly above the cell monolayer in standard assays. The vast majority of Seahorse metabolic studies are performed using in vitro samples due to existing standardized approaches for adherent or suspension cell culture samples. However, the ability to assess ECAR and OCR in ex vivo tissue samples broadens the scope of Seahorse assays to better assess organ-level metabolism, as it pertains to systemic energy utilization and physiology in a manner beyond cell culture.

Several groups have performed Seahorse assays in various tissue types, ranging from intestine to brown and white adipose tissue, the retina, and the hippocampus [1–6]. However, a uniform approach does not exist for using ex vivo tissue samples for Seahorse experiments. Various sample preparation methods have been employed, including using 200–300 µm slices and various size biopsy punches to generate uniform sample sizes with and without tissue mincing [1–6]. Current ex vivo methods have focused primarily on assessing basal oxygen consumption rate and adapting the Mito Stress assay for interrogating mitochondrial function for tissue samples [2–6]. The majority of publications utilized the Agilent XF24 well platform and the corresponding Islet Capture screens, as it provides an established existing framework to hold the tissue samples in place [1–4,6].

In particular, this protocol was designed to measure the extracellular acidification rate of ex vivo murine intestinal samples. In short, our work first identified an increased in vivo glucose uptake from the blood supply to the cystic fibrosis (CF) mouse intestine [1]. Transcriptional and protein-level evidence indicated an enrichment of glycolysis gene expression and increased protein expression of glucose transport and glycolytic enzymes in the CF intestine [1]. This protocol was designed as a functional approach to assess glycolysis in ex vivo intestinal samples. In particular, this approach utilizes the Agilent Seahorse XFe24 platform with Islet Capture microplates and corresponding Islet Capture screens to hold tissue pieces in place.

This ex vivo approach expands beyond the current in vitro methods and allows for functional assessments of metabolism in tissue samples. While this method was specifically designed for assessing extracellular acidification rate in ex vivo murine intestinal samples, the same approach can be applied to other tissue types to interrogate other metabolic processes. For instance, the current method can be modified to assess mitochondrial function through measurements of OCR in numerous tissue types. Overall, this protocol broadens the scope of Seahorse metabolic assays to include tissue samples to establish a framework for better assessing organ-level metabolism in the context of systemic nutrient metabolism and physiology.