PROJECT SUMMARY Just within the past few years, there has been a remarkable progress in the field of single-cell and spatial biology, illustrating the importance of understanding how particular cell types and cell states relate with one another. The Mass Cytometry Facility at Johns Hopkins University (JHU), an operation within the Flow/Mass Cytometry Shared Resource at the Sidney Kimmel Comprehensive Cancer Center (SKCCC), houses just one mass cytometry platform, the Helios/Hyperion system, which switches between suspension and imaging modes. This system is currently the only mass cytometry unit that is accessible by the Johns Hopkins community. Mass cytometry overcomes any issues with fluorescence, such as cellular or tissue autofluorescence and spectral spillover, allowing for the robust assessment of 40+ parameters at a per-cell basis. Since the establishment of mass cytometry services in August 2021 made possible by the support of the Johns Hopkins Convergence Institute and the JHU SKCCC, the usage of mass cytometry has been consistently increasing. With the recent funding of major research activities by the NIH, the demand for mass cytometry services is expected to continue growing dramatically, prompting this proposal for the funds to acquire a second mass cytometer in the form of CyTOF XT, the latest generation mass cytometer, at our Mass Cytometry Facility. Mass cytometry will be incorporated into the NIH-funded research activities led by more than 10 Major and 5 Minor Users across multiple research programs and departments at Johns Hopkins, tackling important questions in pancreatic cancers, liver cancers, breast cancers, computational biology, and dermatology. This unique discovery tool will also support the research of several early-career investigators and other future users from the Johns Hopkins research community at large. The successful implementation of a second mass cytometer will be facilitated by Dr. Won Jin Ho’s (PI) expertise in mass cytometry techniques, experienced facility staff, existing infrastructure for the system, and strong commitments from the School of Medicine, the SKCCC, the Convergence Institute, as well as the Department of Cell Biology, the Department of Pathology, and the Division of Quantitative Sciences at Johns Hopkins. This much needed expansion of mass cytometry services will sustain the prompt delivery of high quality data to the researchers at Johns Hopkins and significantly accelerate the discovery process in biomedical research.