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 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 um above the bottom of the wells, entrapping ~ 7 uL 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, pancreatic islets/beta cells, and isolated mitochondria. Cell samples can be reused after measurements, including determinations of protein, ATP concentration, or cell number by Hoechst staining. Custom plates containing a central well depression and grid are suitable for study of whole islets.
The Seahorse XF24 Extracellular Analyzer is located in J520 on the fifth floor of the biomedical science 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.