Seahorse XF24 Cellular Bioenergetics Analysis

Tuesday 06/01/2010

ATP generation by mitochondrial oxidative phosphorylation and glycolytic metabolism is essential for all energy-requiring processes in the cell, yet bioenergetic capacity and flux are tightly regulated using a just-in-time strategy to meet real or anticipated needs. Metabolic partitioning between glycolysis and oxidative phosphorylation is also subject to biosynthetic requirements, as both pathways generate intermediates for protein, DNA and lipid biosynthesis. Thus, changes in bioenergetic metabolism are coupled to diverse processes such as cell activation, mitosis and differentiation, and measurements of glycolysis and oxidative phosphorylation rates may be informative for both normal physiological and disease states.

Extracellular flux assays measure O2, total acid, and CO2 in cell media and provide accuracies comparable to radiometric assays. Previously, these assays required separate devices, such as Clark electrode chambers for oxymetry, and were not suitable for cells growing in standard tissue culture formats. The Seahorse XF24 instrument was recently developed as a multi-well plate-based assay platform that uses fluorescent optode detectors to measure oxygen consumption rates (OCR) and extracellular acid release (ECAR) from cells plated in custom 24-well plates.  The optodes consist of a pH or O2 sensitive fluorescent dye dispersed in a small plug of hydrogel. The sensor gels are spotted onto a small plunger attached to the plate lid. These plungers normally reside about 5 mm above the cells within about 1 mL of buffer. During a rate measurement, the plungers descend to ~300 m above the bottom of the wells, entrapping ~ 7 L of buffer with limited diffusion to create a “virtual chamber”. Analyte concentrations are measured in each well once every 10-20 s over a period of typically 90-120 s. OCR and ECAR are calculated from the slopes for the change in concentration automatically using integrated software. After rate measurements, the plungers return to their original positions, and vibrate to mix the buffer and re-equilibrate the sampled medium with the bulk medium. Rate measurements can be made every few minutes over periods up to 24 h without significant depression of oxygen tension or acidification of the media.

Cells are assayed in a custom-prepared media lacking CO2/HCO3 or HEPES buffers, and equilibrated with this media for 1 hour prior to starting measurements. The Seahorse instrument maintains plate temperatures at 37°C. Above each well of the Seahorse 24-well plates are 4 cylindrical reagent reservoirs. The software permits programmable injection of each reservoir adding up to 4 different reagents during an experiment. The well size and alignment of the reservoirs matches a standard 96-well plate so that loading can be performed with standard pipettors. Plates and sensor cartridges are disposable to avoid cross-contamination from the sensors between experiments. A variety of established cell lines and primary cell types have been investigated with the Seahorse XF24, including neurons, lymphocytes, and isolated mitochondria. Cell samples can be reused after measurements, including determinations of protein or cell number by Hoechst staining. Custom plates containing a central well depression and grid are suitable for study of whole islets.

Services are an expansion of the Cytokine Analysis Shared Resource.  The Seahorse XF24 Extracellular Analyzer is located in room D2-383 on the second floor of the Thomas Building.  A dedicated room air incubator is kept adjacent to the Analyzer for pre-measurement equilibration.  Experiments are designed using the XF Reader Software on a touch screen, and can be analyzed using Excel spreadsheets.

Dr. David Hockenbery is the Director of this unit, with 20 years of experience in measurements of mitochondrial and bioenergetic metabolism. The unit Supervisor, Dr. Daciana Margineantu, has extensive experience in mitochondrial biology and bioenergetic metabolism, including specialized training on the Seahorse instrument at the company headquarters.  The Lead Research Technician, John Fry, has extensive experience in cell culture, viability and metabolism assays, including design and analysis of experiments using the Seahorse XF24 Analyzer.

An initial meeting with resource staff is highly recommended to plan experiments in accordance with specific research goals. Staff will provide plates and sensor cartridges, prepare custom media, and analyze OCR and ECAR for investigator-provided cell samples. Standard reagents for measurements of mitochondrial coupling efficiency, mitochondrial reserve, respiratory control and substrate fuel utilization are provided. In addition, custom studies using a variety of metabolic inhibitors are possible.  Resource staff will analyze data or provide instruction in using Seahorse software. Training on instrument can also be provided, for unassisted measurements.  Scheduling for the Seahorse instrument can be made up to 2 weeks in advance.