This proposal requests funds for a highly-parametric, user-friendly analytical cytometer to be incorporated into the Research Flow Cytometry Core (RFCC) that serves investigators at Cincinnati Children?s Hospital Medical Center (CCHMC) and University of Cincinnati College of Medicine (UCCOM). The BioRad ZE5 analytical cytometer has been selected as it best fulfills the needs of our NIH funded users. A dramatic expansion of research in the past 10 years at CCHMC and UCCOM has increased the number of investigators utilizing single cell analysis techniques. This is reflected by (1) an increase in the number of investigators using the RFCC analytical cytometers from 119 in 2011, to 191 in 2016 and (2) a consistently high utilization rate of greater than 70% on our high-end instruments that has limited the accessibility of these instruments. In addition, our current high-end analytical cytometers are limited to a maximum of 18 fluorescence parameters for detection and major users are in need of access to higher parameter technology as their projects involve characterization of small sample sizes and require assessment of higher number of cellular markers to delineate specific cell populations important in disease processes. These factors indicate an increased need for (1) a user-friendly instrument that can acquire an increased number of cellular parameters, and (2) a cytometer that can easily acquire samples from a 96-well plate. The instrument will support and enhance existing NIH-funded projects and will foster avenues of research that will likely lead to future funded projects. In this application, 16 investigators (8 major and 8 minor users, with all users funded by their own NIH grants) are identified whose projects require increased accessibility to high multi-parameter cellular analysis. The major users? projects span a broad spectrum of biologic queries requiring the use of single cell analysis, including: (1) tumor-associated immune responses; (2) biology of human regulatory T cells; (3) pathophysiology and treatment of immunoregulatory disorders; (4) transcriptional control of hematopoiesis and cancer; (5) immune mechanisms in treatment of autoimmune diabetes; (6) hematopoietic stem cell proliferation and differentiation; (7) natural killer cell contributions to antiviral immunity and vaccine efficacy (8) fundamental mechanism of hematopoietic stem cell self-renewal. The incorporation of the ZE5 into our current analytical cytometer repertoire will complement and enhance the operation of the RFCC and maximally benefit investigators in this application, as well as the entire user base in the CCHMC and UCCOM research community.