Cellular Bioenergetics Analysis
Location: Thomas building, D2-383
Contact phone: (206) 667-4611
Contact fax: (206) 667-6845
Contact e-mail: firstname.lastname@example.org
Measurements of O2 consumption and acid release are used to assess the bioenergetic state of cells. The recently acquired Seahorse XF24 can be used to analyze the bioenergetic state of cells in standard tissue culture. The Seahorse instrument is 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.
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.
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.