This request is for funds to purchase an Amnis ImageStreamx-markII imaging flow cytometer with 4 lasers for fluorescence excitation (405, 488, 561, 642) and 12 detectors. There is no ImageStream at the University of Pittsburgh or within 100 miles. Unique as an instrument, the ImageStream combines the strengths of flow cytometry (multiplex, quantitative, sensitive) with microscopy (subcellular detail, fluorescence localization, and morphology) to permit the simultaneous multiparameter analysis of cells at the plasma membrane, subcellular, and molecular levels. The instrument will be housed within the Unified Flow Cytometry Core, a centralized core, at the University of Pittsburgh School of Medicine. The Unified Flow Core currently serves over 150 PIs and 500 individual Users. For the proposed instrument, Major Users include 13 PIs (12 NIH funded) with another 7 PIs as Minor Users. Together, these PIs have over $50 M in NIH-funded direct costs from at least 6 different institutes. A major research focus is in the broad area of immunology and allied immune diseases, with sister disciplines such as transplantation, rheumatology, vascular biology, and oncology also well represented. While the Unified Flow Cytometry Core is well-endowed with traditional (though busy) analyzers and sorters, the ImageStream brings totally new breadth to our research capabilities. Our extensive preliminary and demo work proved to us that this instrument will truly allow our investigators to break new ground in their research. The demo phase of this instrument indeed created great excitement among the PIs and hands-on Users. The new machine will be well cared for by the staff of the Unified Flow Cytometry Core, which has an extensive infrastructure and culture in place to train Users, oversee the machine and ensure that it is self-supporting. Two different core personnel will be responsible for the conceptual and day to day aspects of experiments on the ImageStream. We will start and certify the machine each morning and troubleshoot any problems, as we will do at any time during working hours. As such, it will have a remarkable impact on the productivity of an outstanding and well-funded cohort of PHS investigators.
