The project is a ShEEP request for a Molecular Devices SpectraMax i3x Multi-Mode Microplate Reader to replace a non-functional microplate reader in the research core laboratories of the Baltimore VA Medical Center. The SpectraMax i3x Multi-Mode microplate reader measures spectral-based absorbance, fluorescence, and luminescence with the added functionality of modular upgrades for: western blot, cellular imaging, and fast kinetics with injectors. It allows users to explore cellular pathways, live cell imaging, and protein activation and expression with greater ease. The microplate reader offers expanded dynamic range and user-upgraded applications. The instrument will facilitate individual VA investigators with widely varying projects to perform a variety of laboratory experiments including: immunoassays, enzymatic assays, quantification of proteins and nucleic acids, as well as live cell imaging. The requested instrument will support the work of the entire Baltimore VA Medical Center Research Service, including nine major VA Merit-funded users (Drs. Hornyak, Atamas, Carrier, Jaladanki, Kristian, Luzina, Tonelli, Wang, and Xie), and one minor user (Dr. Turner). Thomas J. Hornyak, M.D., Ph.D., Associate Chief Of Staff (ACOS) for Research and Development (R&D) at the VA Medical Health Center System (VAMHCS) is the Principal Investigator (PI) for this proposal. He is also PI on the Merit Review project ?Melanocyte Stem Cells in Regenerative Medicine?. He and his laboratory will use the microplate reader to evaluate relative levels of expression of recently identified genetic signatures in melanocytic and neural crest-like cell lines. Sergei Atamas, M.D., Ph.D. and Irina Luzina, M.D., Ph.D., are individually funded VA Merit Review Investigators who collaborate on the study of interleukin (IL)-33 and pulmonary fibrosis. Their laboratory groups will use the microplate reader to analyze immunoassays and western blots. They will also use the imaging cytometer to assess the nuclear and cytoplasmic localization of recombinant proteins in cells transfected with various recombinant constructs. France Carrier, Ph.D., is a VA Merit Funded investigator whose VA Merit project focuses on mechanisms of chemopotentiation by Low Dose Fractionated Radiation Therapy for disseminated intra-abdominal cancers. Her laboratory will use the microplate reader to measure levels of Reactive Oxygen Species, protein carbonylation, and phosphorylation of IkB. Rao Jaladanki, Ph.D., Douglas Turner, M.D., and Jian-Ying Wang, M.D., Ph.D., are individually funded VA Merit Review Investigators whose research programs are focused on gut mucosal healing and homeostasis. Their studies will utilize the microplate reader to quantitatively assay protein content, for polyamine assays, FITC-Dextran uptake measurements, and other routine spectrophotometric quantitation in extracted samples from: in vitro cell culture samples, human samples and mouse intestinal mucosal scrapings. Tibor Kristian, Ph.D. is a funded VA Merit Investigator whose research focuses on understanding the molecular processes involved in acute brain injury. His laboratory will utilize the microplate reader for determining the levels of metabolites such as Nicotinamide adenine dinucleotide (NAD), and pyrophosphate in brain samples. Leonardo Tonelli, Ph.D. has a funded VA Merit project that studies the relation between systemic inflammation and emotional dysregulation in patients who suffer from depression, anxiety disorders, and post-traumatic stress disorder. His laboratory will use the microplate reader to identify small proteins that CD4+ cells with protective function may be producing after exposure to traumatic stress. Guofeng Xie, MD's VA-funded work focuses on delineating molecular mechanisms behind colon cancer progression. He will use the SpectraMax i3X microplate reader to accurately measure luciferase activity in transfected human colon cancer cell lines so that his laboratory can determine the Farnesoid X Receptor (FXR)-responsive element in the Matrix metalloproteinase 7 (MMP7) gene promoter.
The broad, long-term objectives of this application are to provide a specialized research instrument to a group of VA research investigators who are conducting VA-funded studies on problems that are relevant to Veteran health. The Molecular Devices SpectraMax i3x Multi-Mode Microplate Detection Platform is a user-friendly instrument that will be housed within the laboratory space of the Baltimore VA Medical Center; and will be available for use by VA investigators pursuing projects in many areas including: cancer research, inflammation and lung fibrosis, gut mucosal healing, and acute brain injury. The SpectraMax i3x Microplate Reader will support a wide range of laboratory experiments including: immunoassays, quantification of proteins and nucleic acids, as well as live cell imaging. The instrument will also help stimulate collaborations between these investigators through sharing of data and technical expertise.