Project Summary Vanderbilt University is requesting funds for an Agilent Seahorse XF Pro Analyzer. The Seahorse Analyzer allows the non-invasive real-time study of cellular bioenergetics in adherent or suspension cells, permeabilized cells, ex vivo tissue, isolated mitochondria, organoids, or whole organisms. A previous S10-funded Seahorse XFe96 is housed and operated in the Vanderbilt High-throughput Screening (VHTS) Facility for the past 9 years. Current use of the instrument over the past 5 years has averaged- 450 annual hours and ~40 users from 30 funded projects. A very broad spectrum of fields of research have used the instrument, including investigators from 40 different departments at Vanderbilt University and Medical Center. The major areas of interest include numerous disease states in which metabolic dysfunction has been implicated, such as cancer, obesity, immunology, cardiology, and neurodegenerative disorders. The instrument use has resulted in 33 publications, to date. Clearly the need for continuing these bioenergetic studies at Vanderbilt is critical to many areas of biomedical research. Although the XFe96 Analyzer has been an outstanding instrument, it is past its life expectancy, and critical components have had to be replaced in the past few years, including the main computer board. Thus, it is crucial that this instrument is replaced as soon as possible to avoid delays or halting of NIH- funded projects that aim to advancing their bioenergetics studies in basic or early translational research. The proposed upgrade will be the newest Agilent Seahorse Analyzer model, the XF Pro. This model significantly improves the sensitivity, temperature control, reduces plate and sample variability, and has upgraded software to better integrate plate annotations, data normalization, analyses, and multiplexed assays. The XF Pro will be integrated for automated cell normalization through an accompanying plate imaging system to provide seamless workflows and quick, robust data analyses. The upgraded Seahorse Analyzer will continue to enhance current and future research in bioenergetics at Vanderbilt.