Project Summary/Abstract Quantifying the presence, function, and fate of different immune cells is crucial in immunology and immuno- oncology research, as well as in the diagnosis of hematologic disorders. Flow cytometry is the current method of choice to measure different proteins expressed by single cells at high throughput. However, spectral overlap between fluorophore labels makes flow measurement of more than 15 protein markers difficult, often requiring months of optimization, while measuring more than 40 markers is not possible with current technologies. This upper bound prevents more sophisticated analysis of cell state and function and limits discovery of immune biomarkers needed for treatment monitoring and prognostication. Other methods for high-marker analysis including mass cytometry and RNA-seq based methods are severely limited in throughput and cost-prohibitive, and not ideal for measuring patient blood samples containing millions of cells. The long-term goal of this application is to develop cyclic flow cytometry, a high-throughput, low cost solution for high-marker single-cell analysis. Cyclic flow cytometry leverages proprietary laser particle (LP) technology to barcode individual cells for repeated flow measurements and, once fully developed, is expected to be capable of measuring unprecedented 100 markers at high throughput. The specific aims of this Phase I involve developing key processes required for cyclic cytometry. We will optimize the process of LP delivery into human peripheral mononuclear cells (PBMCs) and will build a flow prototype device for measuring laser and fluorescence emission simultaneously and validating single-cell tracking over multiple flow cycles. Completion of Phase I research will establish the feasibility of cyclic flow cytometry. Full integration of the entire processes and demonstration of 100 marker analysis will be the subject of a Phase II submission.